http://compbio.biosci.uq.edu.au/mediawiki/api.php?action=feedcontributions&user=Basma&feedformat=atomMDWiki - User contributions [en]2024-03-28T21:15:36ZUser contributionsMediaWiki 1.39.6http://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10825Arylformamidase Results2008-06-10T02:05:43Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Regions of sequence were identified which were highly conserved between prokaryotic and eukaryotic species (see figure 7a). These residues were annotated on the structure of 2PBL (see figure 7b). The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Alignment1.jpg|centre|framed|'''Figure 7a:''' ''The multiple sequence alignment performed between prokaryotic and eukaryotic sequences showing conserved regions.'']]<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7b:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10823Arylformamidase Results2008-06-10T02:04:28Z<p>Basma: /* Structure */</p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Regions of sequence were identified which were highly conserved between prokaryotic and eukaryotic species (see figure 7a). These residues were annotated on the structure of 2PBL (see figure 7b). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Alignment1.jpg|centre|framed|'''Figure 7a:''' ''The multiple sequence alignment performed between prokaryotic and eukaryotic sequences showing conserved regions.'']]<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7b:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10821Arylformamidase Results2008-06-10T02:02:38Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
[[Image:Alignment1.jpg|centre|framed|'''Figure 7b:''' ''The multiple sequence alignment performed between prokaryotic and eukaryotic sequences showing conserved regions.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10817Arylformamidase Results2008-06-10T02:01:55Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Image:Alignment1.jpg|centre|framed|'''Figure 7b:''' ''The multiple sequence alignment performed between prokaryotic and eukaryotic sequences showing conserved regions.'']]<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10814Arylformamidase Results2008-06-10T02:01:15Z<p>Basma: /* Structure */</p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
[[Image:Alignment1.jpg|centre|framed|'''Figure 7b:''' ''The multiple sequence alignment performed between prokaryotic and eukaryotic sequences showing conserved regions.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Function_Slide_6&diff=10805Arylformamidase Function Slide 62008-06-10T01:57:55Z<p>Basma: </p>
<hr />
<div>== Similarity in mechanism? ==<br />
<br />
[[Image:Arylformadisae_reaction.gif|centre|framed|The reaction catalysed by Arylformamidase.]]<br />
[[Image:Carboxylesterase_reaction.gif|centre|framed|The fundamental reaction catalysed by carboxylesterases.]]<br />
<br />
- Both involve hydrolysis.<br />
<br />
- May be A/B hydrolase.<br />
<br />
- Prokaryotic evidence stronger.<br />
<br />
[[Arylformamidase Function Slide 5| ...Previous slide ]]|[[Arylformamidase| Return to the main page ]]|[[Arylformamidase Function Slide 7| Next slide... ]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Function_Slide_6&diff=10797Arylformamidase Function Slide 62008-06-10T01:55:36Z<p>Basma: </p>
<hr />
<div>== Similarity in mechanism? ==<br />
<br />
[[Image:Arylformadisae_reaction.gif|centre|framed|The reaction catalysed by Arylformamidase.]]<br />
[[Image:Carboxylesterase_reaction.gif|centre|framed|The fundamental reaction catalysed by carboxylesterases.]]<br />
<br />
- Both involve hydrolysis.<br />
<br />
- May be A/B hydrolase.<br />
<br />
[[Arylformamidase Function Slide 5| ...Previous slide ]]|[[Arylformamidase| Return to the main page ]]|[[Arylformamidase Function Slide 7| Next slide... ]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Discussion&diff=10795Arylformamidase Discussion2008-06-10T01:54:20Z<p>Basma: </p>
<hr />
<div>Functional inference from sequence similarity was restricted due to lack of supporting literature for highest-scoring sequences. Although the function of the arylformamidase from ''Mus musculus'' has been relatively well-characterised (Pabarcus 2005), its structure has not been determined. Arylformamidase proteins have not been characterised from amongst the prokaryotes, indicating it may be limited to the eukaryotes. Whilst a high-degree of conservation of the catalytic triad has been observed, there are proteins more structurally similar which provides a greater indication of function. <br />
<br />
The carboxylesterase 2C7B was chosen for further analysis due to its high structural similarity to 2PBL. 2C7B is a thermostable carboxylesterase from a metagenomic archaeon which was isolated in a thermal environmental sample (Byun 2007). This carboxylesterase belongs to a family of bacterial hormone-sensitive lipases/esterases (HSLs). There were a number of other bacterial HSL proteins as identified by Byun, et al. (2007) found to have a high structural similarity to 2PBL. Although a relatively low sequence similarity with 2PBL was observed, regions of conserved sequence characteristic to HSL proteins aligned with 2PBL (Arpigny 1999). This may indicate that 2PBL is a member of the bacterial HSL family of lipolytic proteins. The HSL family encompasses a broad range of proteins with relatively diverse functionality which are known for their amino-acid sequence similarity to the mammalian hormone-sensitive lipase (Arpigny 1999). Contrary to this, a mammalian hormone-sensitive lipase was not returned in the BLAST search, significant evidence against 2C7B being a member of the HSL family. <br />
<br />
Further analysis revealed that 2PBL shares greater structural similarity with thermostable esterases of the HSL family compared to its other members. As discussed, thermostable proteins such as 2C7B often exist in dimers, a possiblity which remains uninvestigated for 2PBL. Both 2PBL and 2C7B have been hypothesised to exist functionally as dimers, a feature characteristic of thermostable proteins (Byun, et al. 2007). By examining the crystal structure and sequence homology, we have attempted to deduce the functional form of 2PBL. It is expected that our protein exists as a monomer indicating that it may not have thermostable properties, but this remains to be experimentally confirmed.<br />
<br />
Clues to the function of 2PBL may be inferred from similarities between the fundamental reaction mechanisms of arylformamidases and carboxylesterases. Arylformamidase catalyses the hydrolysis of N-formyl-L-kynurenine to L-kynurenine, the second step in the conversion of tryptophan to nicotinic acid, NAD(H) and NADP(H) (see figure 16). Carboxylesterases catalyse the hydrolysis of a carboxylic ester into an alcohol and a carboxylic acid (see figure 17). Indeed, both arylformamidase and carboxylesterase are members of the A/B-hydrolase superfamily, suggesting that 2PBL is a A/B-hydrolase. <br />
<br />
[[Image:Arylformadisae_reaction.gif|centre|framed|'''Figure 16:''' ''Reaction mechanism of the mammalian arylformamidase. Produced using ChemSketch.'']]<br />
<br />
[[Image:carboxylesterase_reaction.png|centre|framed|'''Figure 17:''' ''Fundamental reaction mechanism of carboxylesterases.'']]<br />
<br />
<br />
A multiple sequence alignment revealed several conserved regions in the sequence of 2PBL across all species. This high level of conservation was noted from the Bacteria through to the Eukaryota including vertebrates, invertebrates, yeasts and moulds. Most significantly, a relatively conserved catalytic triad consisting of serine, aspartimic/glutamic acid and histidine as well as associated residues was observed in many of these sequences. The group of catalytic residues occurred in the putative functional region of 2PBL. This may be indicative of a protein scaffold used ubiquitously in the evolution of hydrolases.<br />
<br />
The phylogeny of our protein is largely consistent with traditional taxonomic groupings of organisms. Also, from the cladistics of the phylogenetic tree, there is no evidence for horizontal gene transfer (see figure ...). Thus, the delineations between prokaryotic and eukaryotic species allow us to infer that the dominant mode of inheritance of this functional region is clonal from bacteria to plantae and animalia.<br />
<br />
The A/B-hydrolase superfamily encompasses proteins with a broad range of functions. We have attempted to identify function through analysis of eukaryotic homologues, however a greater understanding of its prokaryotic function remains unclear. Identifying a specific function for 2PBL is a challenge for future research. <br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Discussion&diff=10794Arylformamidase Discussion2008-06-10T01:54:04Z<p>Basma: </p>
<hr />
<div>Functional inference from sequence similarity was restricted due to lack of supporting literature for highest-scoring sequences. Although the function of the arylformamidase from ''Mus musculus'' has been relatively well-characterised (Pabarcus 2005), its structure has not been determined. Arylformamidase proteins have not been characterised from amongst the prokaryotes, indicating it may be limited to the eukaryotes. Whilst a high-degree of conservation of the catalytic triad has been observed, there are proteins more structurally similar which provides a greater indication of function. <br />
<br />
The carboxylesterase 2C7B was chosen for further analysis due to its high structural similarity to 2PBL. 2C7B is a thermostable carboxylesterase from a metagenomic archaeon which was isolated in a thermal environmental sample (Byun 2007). This carboxylesterase belongs to a family of bacterial hormone-sensitive lipases/esterases (HSLs). There were a number of other bacterial HSL proteins as identified by Byun, et al. (2007) found to have a high structural similarity to 2PBL. Although a relatively low sequence similarity with 2PBL was observed, regions of conserved sequence characteristic to HSL proteins aligned with 2PBL (Arpigny 1999). This may indicate that 2PBL is a member of the bacterial HSL family of lipolytic proteins. The HSL family encompasses a broad range of proteins with relatively diverse functionality which are known for their amino-acid sequence similarity to the mammalian hormone-sensitive lipase (Arpigny 1999). Contrary to this, a mammalian hormone-sensitive lipase was not returned in the BLAST search, significant evidence against 2C7B being a member of the HSL family. <br />
<br />
Further analysis revealed that 2PBL shares greater structural similarity with thermostable esterases of the HSL family compared to its other members. As discussed, thermostable proteins such as 2C7B often exist in dimers, a possiblity which remains uninvestigated for 2PBL. Both 2PBL and 2C7B have been hypothesised to exist functionally as dimers, a feature characteristic of thermostable proteins (Byun, et al. 2007). By examining the crystal structure and sequence homology, we have attempted to deduce the functional form of 2PBL. It is expected that our protein exists as a monomer indicating that it may not have thermostable properties, but this remains to be experimentally confirmed.<br />
<br />
Clues to the function of 2PBL may be inferred from similarities between the fundamental reaction mechanisms of arylformamidases and carboxylesterases. Arylformamidase catalyses the hydrolysis of N-formyl-L-kynurenine to L-kynurenine, the second step in the conversion of tryptophan to nicotinic acid, NAD(H) and NADP(H) (see figure 16). Carboxylesterases catalyse the hydrolysis of a carboxylic ester into an alcohol and a carboxylic acid (see figure 1). Indeed, both arylformamidase and carboxylesterase are members of the A/B-hydrolase superfamily, suggesting that 2PBL is a A/B-hydrolase. <br />
<br />
[[Image:Arylformadisae_reaction.gif|centre|framed|'''Figure 16:''' ''Reaction mechanism of the mammalian arylformamidase. Produced using ChemSketch.'']]<br />
<br />
[[Image:carboxylesterase_reaction.png|centre|framed|'''Figure 17:''' ''Fundamental reaction mechanism of carboxylesterases.'']]<br />
<br />
<br />
A multiple sequence alignment revealed several conserved regions in the sequence of 2PBL across all species. This high level of conservation was noted from the Bacteria through to the Eukaryota including vertebrates, invertebrates, yeasts and moulds. Most significantly, a relatively conserved catalytic triad consisting of serine, aspartimic/glutamic acid and histidine as well as associated residues was observed in many of these sequences. The group of catalytic residues occurred in the putative functional region of 2PBL. This may be indicative of a protein scaffold used ubiquitously in the evolution of hydrolases.<br />
<br />
The phylogeny of our protein is largely consistent with traditional taxonomic groupings of organisms. Also, from the cladistics of the phylogenetic tree, there is no evidence for horizontal gene transfer (see figure ...). Thus, the delineations between prokaryotic and eukaryotic species allow us to infer that the dominant mode of inheritance of this functional region is clonal from bacteria to plantae and animalia.<br />
<br />
The A/B-hydrolase superfamily encompasses proteins with a broad range of functions. We have attempted to identify function through analysis of eukaryotic homologues, however a greater understanding of its prokaryotic function remains unclear. Identifying a specific function for 2PBL is a challenge for future research. <br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10791Arylformamidase Results2008-06-10T01:52:54Z<p>Basma: /* Sequence & Homology */</p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure 14. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure 14:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure 15). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure 15:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10790Arylformamidase Results2008-06-10T01:52:23Z<p>Basma: /* Function */</p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure 8). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure 9). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure 10).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Figure 8: ''Selected results of the BLAST search as performed by Sebastien.'']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 9:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 10:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure 11). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure 12). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure 11''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure 12:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure 11). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure 12). <br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 12:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure .... The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure ...). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=10780Arylformamidase Results2008-06-10T01:49:41Z<p>Basma: /* Structure */</p>
<hr />
<div>== Structure ==<br />
<br />
'''2PBL Biological Structure'''<br />
<br />
The functional biological structure of 2PBL is assumed by PDB to be a monomer (see figure 3) even though the 'whole' protein is shown to be interacting with chains A, B, C and D. <br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 3:''' ''2PBL exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
'''2PBL Structural Similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to be mostly carboxylesterases/hydrolases. The first significant hit from DALI was a carboxylesterase from a metagenomic Archeaon. The second significant hit was a carboxylesterase of ''Archaeoglobus fulgidus''. Analysis of 2PBL secondary structure similarity with the Archeal carboxylesterases showed conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix (see figures 4 & 5).<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 4:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 5:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The ClustalW alignment showed conservation of the Archeal carboxylesterases catalytic triad in 2PBL. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 6:''' ''The putative catalytic triad of 2PBL encompassing Serine 136, Histidine 241 and Glutamate 214. The unknown ligand (blue) protrudes from a surface groove. Image generated using Pymol.'']]<br />
<br />
Residues that were highly conserved between prokaryotic and eukaryotic species were annotated on the structure of 2PBL (see figure...). Distances between the conserved residues of the catalytic triad were measured. The blue region in the figure below shows the clustering of conserved residues around the unknown ligand. <br />
<br />
'''Conserved''' - Asp53, His69, Gly70, Gly71, Trp73, Gly134, Ser136, Ala137, Gly138, His241.<br />
'''Semi-conserved''' - Tyr72, His140, Ser166, Leu168, Leu171, Leu174, Glu214, Val244, Leu248.<br />
'''Catalytic triad''' - Ser136, Glu214, His241.<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 7:''' ''The conserved residues annotated on the structure of 2PBL. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment. Image generated using Pymol.'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence from the BLAST search with functional information available was an arylformamidase isolated from the liver of ''Mus musculus'' (see figure ...). A functional analysis of this arylformamidase has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2PBL was assessed using sequence homology (see figure...). Both residues Ser162 and His279 were found to be identical in relatively conserved regions of the alignment. However, Asp247 had undergone a semi-conservative substitution to glutamic acid. These residues correlated to Ser136, Glu214 and His241 of 2PBL which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:2pblBLAST.png|centre|framed|'''Selected results of the BLAST search as performed by Sebastien.''']]<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure ...:''' ''Conservation of the catalytic triad between Arylformamidase and 2PBL.'']]<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ...:''' ''The putative catalytic triad identified through conservation with arylformamidase from ''Mus musculus''. Distances between the residues are shown. Note how each amino acid is linked to a turn region in the amino acid backbone. Generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
2PBL was found to share most structural similarity with a thermostable carboxylesterase from a metagenomic archaeon (PDB ID: 2C7B; see figure ...). 2C7B shares a 16% sequence identity with 2PBL. From its structure, a catalytic triad has been identified with the residues Ser154, Asp251 and His281 (Byun, et al. 2007). To substantiate any functional similarity between 2PBL and 2C7B, structural conservation of the 2C7B catalytic triad was analysed (see figure ...). Both Ser154 and His281 matched, but the aspartic acid had undergone a semi-conservative substitution to glutamic acid. <br />
<br />
[[Image:2c7b_alignment2.png|centre|framed|'''Figure ...:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
A number of proteins from the hormone-sensitive lipase (HSL) class of lipolytic enzymes as identified by Byun, et al. (2007) was found within top-scoring results of the DALI search (see figure...). To characterise sequence similarity, a multiple sequence alignment of the amino acid sequences for these structures was performed (see figure ...). <br />
<br />
[[Image:DAL_HSLs.png|centre|framed|'''Figure ...''' ''Selected results of the DALI search performed by Basma encompassing structures of the HSL family of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure ...:''' ''Alignment with members of the HSL class of lipolytic enzymes as identified by Byun, et al. (2007).'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
The phylogenetic tree constructed using the multiple sequence alignment on 2PBL related sequences is shown in figure .... The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree. This reveals a possible explanation for certain closely related species to be grouped into separate clades. Despite low bootstrap scores, the grouping reliably separates prokaryotes from eukaryotes. Interestingly, homologous eukaryotes include yeasts and moulds, plants, invertebrates and vertebrates.<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter species'' from which 2PBL originated. The colour coding distinguishes prokaryotic organisms shown in blue, from eukaryote yeasts and moulds (shown in green), plants (dark green), invertebrates (orange) and vertebrates (shown in red).'']]<br />
<br />
To further elucidate the phylogeny of 2pbl, its human homologue, an arylformamidase, was queried in a BLAST search. The top scoring matches of bacterial homologues, present in figure 1, were appended with top scoring matches of eukaryotic homologues. The human homologue has a 26.28% sequence similarity. Despite this low score, multiple sequence alignment revealed that key regions were highly conserved between bacterial and eukaryotic homologues. This was demonstrated in a phylogenetic analysis of 2PBL and its human homologue which was largely consistent with traditional taxonomic groupings of organisms(see figure ...). Specifically, it reveals greater statistical confidence in the separation of prokaryotes and eukaryotes.<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the ''Silicibacter'' species from which 2PBL originated. The colour coding distinguishes prokaryotes (blue and green) and eukaryotes (invertebrates are shown in orange; vertebrates are in red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. Notably, members of the ''Silicibacter'' clade occur on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Additional_Materials&diff=10747Arylformamidase Additional Materials2008-06-10T01:36:21Z<p>Basma: </p>
<hr />
<div>'''Links'''<br />
<br />
[http://www.pdb.org/pdb/explore.do?structureId=2PBL Protein Data Bank Entry for 2PBL]<br />
<br />
'''FASTA Sequence'''<br />
<br />
>gi|146387357|pdb|2PBL|A Chain A, Crystal Structure Of Putative Thioesterase (Yp_614486.1) From Silicibacter Sp. Tm1040 At 1.79 A Resolution<br />
GXELDDAYANGAYIEGAADYPPRWAASAEDFRNSLQDRARLNLSYGEGDRHKFDLFLPEGTPVGLFVFVH<br />
GGYWXAFDKSSWSHLAVGALSKGWAVAXPSYELCPEVRISEITQQISQAVTAAAKEIDGPIVLAGHSAGG<br />
HLVARXLDPEVLPEAVGARIRNVVPISPLSDLRPLLRTSXNEKFKXDADAAIAESPVEXQNRYDAKVTVW<br />
VGGAERPAFLDQAIWLVEAWDADHVIAFEKHHFNVIEPLADPESDLVAVITA<br />
<br />
[[Arylformamidase | Return to the main page...]]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
Human arylformamidase has the following catalytic activity.<br />
<br />
<br />
<br />
N-formyl-L-kynurenine + H2O → Formate + L-kynurenine <br />
<br />
Required for elimination of toxic metabolites<br />
<br />
It belongs to the AB hydrolase super family.<br />
<br />
Even though the structure of the human protein hasn’t been determined, looking at the sequence alignment the catalytic triad residues are conserved. The residues have resisted mutation indicating that they are important for activity.<br />
<br />
Our protein is similar to carboxylesterase which also belongs to AB suberfamily.<br />
<br />
[[Image:ChainA 2c7b.PNG|left|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. The structure of carboxylesterase shows absence of ligands. From PDB ProteinWorkshop 1.5'']][[Image:ChainA 1jji.PNG|right|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. The ligand is present in this figure. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
Each of the residues are linked to a turn region. The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
'''Sequence alignment:'''<br />
<br />
''Sections of alignment showing the conserved residues across bacterial and eukaryotic species.''<br />
<br />
[[Image:Alignment1.jpg]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Structure&diff=10741Arylformamidase Structure2008-06-10T01:35:29Z<p>Basma: </p>
<hr />
<div>=='''Methods'''==<br />
<br />
The structure of arylfromamidase was obtained from RCSB Protein Data Bank (PDB ID: 2PBL).<br />
http://www.rcsb.org/pdb/home/home.do<br />
<br />
The predicted interaction arylformamidase with other proteins was determined using the STRING database (STRING: Search Tool for the Retrieval of Interacting Genes/Proteins). http://string.embl.de// <br />
<br />
The DALI database was used for the structural comparison of arylformamidase with other proteins. http://ekhidna.biocenter.helsinki.fi/dali_server/<br />
<br />
PDBsum database was used to determine the secondary structure of arylformamidase.<br />
http://www.ebi.ac.uk/pdbsum/<br />
<br />
<br />
<br />
== '''Results''' ==<br />
<br />
<br />
== Structure of Arylformamidase ==<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
<br />
<br />
<br />
'''Figure: Arylformamidase (All Chains)'''<br />
<br />
<br />
[[Image:Arylformamidase the whole protein.PNG]] <br />
<br />
<br />
''The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure:''Chain A'' of arylformamidase'''<br />
<br />
<br />
[[Image:ChainA1.PNG]] <br />
<br />
<br />
''The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5'' <br />
<br />
'''''The protein backbone is coloured by conformation type:'''''<br />
<br />
'''''Turn - blue'''''<br />
<br />
'''''Coil- pink'''''<br />
<br />
'''''Helix- green'''''<br />
<br />
'''''strand- purple'''''<br />
<br />
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Confidence_interaction_with_names.png]]<br />
<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
== '''Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Examplec.jpg]]<br />
<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
== '''DALI OUTPUT''' ==<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
<br />
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 2c7b.PNG]] <br />
<br />
[[Image:Carboxylase.txt ]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 1jji.PNG]] <br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]<br />
<br />
''Note: Chains B, C & D not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
== '''Secondary structure analysis''' ==<br />
<br />
<br />
<br />
'''PDBSum output for arylformamidase'''<br />
<br />
[[Image:PDBSum pblA.PNG]] <br />
<br />
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]<br />
<br />
<br />
'''Figure: Archeon Carboxylesterase secondary structure'''<br />
<br />
[[Image:Pdbsums archeal.PNG]] <br />
<br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
== '''The conservation of the ser/his/asp catalytic triad''' ==<br />
<br />
[[Image:Catalytic triad conversation.PNG]]<br />
<br />
''Yellow indicates conservation''<br />
<br />
''Blue indicates semi-conservation''<br />
<br />
<br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:Untitled2.PNG]]<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The conserved residues of arylformamidase'''<br />
<br />
<br />
[[Image:Cat triad red.PNG]] <br />
<br />
<br />
''The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG]]<br />
<br />
''The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)''<br />
<br />
'' From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''<br />
<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG]]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
<br />
<br />
'''Sequence alignment:'''<br />
<br />
''Sections of alignment showing the conserved residues across bacterial and eukaryotic species.''<br />
<br />
[[Image:Alignment1.jpg]]<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=File:Alignment1.jpg&diff=10731File:Alignment1.jpg2008-06-10T01:33:43Z<p>Basma: </p>
<hr />
<div></div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Structure&diff=10729Arylformamidase Structure2008-06-10T01:33:30Z<p>Basma: </p>
<hr />
<div>=='''Methods'''==<br />
<br />
The structure of arylfromamidase was obtained from RCSB Protein Data Bank (PDB ID: 2PBL).<br />
http://www.rcsb.org/pdb/home/home.do<br />
<br />
The predicted interaction arylformamidase with other proteins was determined using the STRING database (STRING: Search Tool for the Retrieval of Interacting Genes/Proteins). http://string.embl.de// <br />
<br />
The DALI database was used for the structural comparison of arylformamidase with other proteins. http://ekhidna.biocenter.helsinki.fi/dali_server/<br />
<br />
PDBsum database was used to determine the secondary structure of arylformamidase.<br />
http://www.ebi.ac.uk/pdbsum/<br />
<br />
<br />
<br />
== '''Results''' ==<br />
<br />
<br />
== Structure of Arylformamidase ==<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
<br />
<br />
<br />
'''Figure: Arylformamidase (All Chains)'''<br />
<br />
<br />
[[Image:Arylformamidase the whole protein.PNG]] <br />
<br />
<br />
''The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure:''Chain A'' of arylformamidase'''<br />
<br />
<br />
[[Image:ChainA1.PNG]] <br />
<br />
<br />
''The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5'' <br />
<br />
'''''The protein backbone is coloured by conformation type:'''''<br />
<br />
'''''Turn - blue'''''<br />
<br />
'''''Coil- pink'''''<br />
<br />
'''''Helix- green'''''<br />
<br />
'''''strand- purple'''''<br />
<br />
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Confidence_interaction_with_names.png]]<br />
<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
== '''Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Examplec.jpg]]<br />
<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
== '''DALI OUTPUT''' ==<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
<br />
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 2c7b.PNG]] <br />
<br />
[[Image:Carboxylase.txt ]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 1jji.PNG]] <br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]<br />
<br />
''Note: Chains B, C & D not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
== '''Secondary structure analysis''' ==<br />
<br />
<br />
<br />
'''PDBSum output for arylformamidase'''<br />
<br />
[[Image:PDBSum pblA.PNG]] <br />
<br />
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]<br />
<br />
<br />
'''Figure: Archeon Carboxylesterase secondary structure'''<br />
<br />
[[Image:Pdbsums archeal.PNG]] <br />
<br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
== '''The conservation of the ser/his/asp catalytic triad''' ==<br />
<br />
[[Image:Catalytic triad conversation.PNG]]<br />
<br />
''Yellow indicates conservation''<br />
<br />
''Blue indicates semi-conservation''<br />
<br />
<br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:Untitled2.PNG]]<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The conserved residues of arylformamidase'''<br />
<br />
<br />
[[Image:Cat triad red.PNG]] <br />
<br />
<br />
''The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG]]<br />
<br />
''The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)''<br />
<br />
'' From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''<br />
<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG]]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
<br />
<br />
'''Sequence alignment:'''<br />
<br />
[[Image:Alignment1.jpg]]<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Additional_Materials&diff=10432Arylformamidase Additional Materials2008-06-09T23:41:31Z<p>Basma: </p>
<hr />
<div>'''Links'''<br />
<br />
[http://www.pdb.org/pdb/explore.do?structureId=2PBL Protein Data Bank Entry for 2PBL]<br />
<br />
'''FASTA Sequence'''<br />
<br />
>gi|146387357|pdb|2PBL|A Chain A, Crystal Structure Of Putative Thioesterase (Yp_614486.1) From Silicibacter Sp. Tm1040 At 1.79 A Resolution<br />
GXELDDAYANGAYIEGAADYPPRWAASAEDFRNSLQDRARLNLSYGEGDRHKFDLFLPEGTPVGLFVFVH<br />
GGYWXAFDKSSWSHLAVGALSKGWAVAXPSYELCPEVRISEITQQISQAVTAAAKEIDGPIVLAGHSAGG<br />
HLVARXLDPEVLPEAVGARIRNVVPISPLSDLRPLLRTSXNEKFKXDADAAIAESPVEXQNRYDAKVTVW<br />
VGGAERPAFLDQAIWLVEAWDADHVIAFEKHHFNVIEPLADPESDLVAVITA<br />
<br />
[[Arylformamidase | Return to the main page...]]<br />
<br />
<br />
<br />
Human arylformamidase has the following catalytic activity.<br />
<br />
<br />
N-formyl-L-kynurenine + H2O → Formate + L-kynurenine <br />
<br />
Required for elimination of toxic metabolites<br />
<br />
It belongs to the AB hydrolase super family.<br />
<br />
Even though the structure of the human protein hasn’t been determined, looking at the sequence alignment the catalytic triad residues are conserved. The residues have resisted mutation indicating that they are important for activity.<br />
<br />
Our protein is similar to carboxylesterase which also belongs to AB suberfamily.</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Introduction&diff=10385Arylformamidase Introduction2008-06-09T23:05:37Z<p>Basma: </p>
<hr />
<div>The structure 2pbl, as denoted by its Protein Data Bank (PDB) accession number, was determined using x-ray crystallography at the Joint Center for Structural Genomics (JCSG) (see figure 2). 2pbl heralds from ''Silicibacter sp.'' TM1040, a member of the ''Roseobacter'' clade of alpha-proteobacteria.. One of the goals of the JCSG is to achieve structural coverage of broad range of protein families by analysing homologous sequences from a variety of model organisms including Silicibacter sp. For structural determination, the protein was expressed in ''Escherichia coli'' using a plasmid as the vector. A resolution of 1.79A was achieved with an R-value of 0.224 and an R-free value of 0.270, ''all of which are indiciative of a high-quality structure''. <br />
<br />
[[Image:Arylformamidase the whole protein.PNG|centre|framed|'''Figure 2:''' ''An overview of 2pbl exhibiting all chains: A - upper right, B - upper left, C - lower right & D - lower left. The red molecule in the chain structure is an unknown ligand. The molecules in the middle of chains A & B and chains C & D is a phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). ''Image generated using PDB ProteinWorkshop 1.5.'']]<br />
<br />
Upon crystallisation, 2pbl formed a tetramer structure. However, structures formed upon crystalisation do not always denote the functional form of a protein which can exist as a dimer or oligomer as well. In fact, such forms may have been evolutionarily selected for to confer features such as thermostability (Byun 2007). By examining the crystal structure and sequence homology , we have attempted to deduce the functional form of 2pbl.<br />
<br />
''Silicibacter sp. TM1040'' was first isolated as part of an investigation into the role of bacteria in the physiology and toxigenesis of the dinoflagellate Pfiestera piscicida. Silicibacter sp. TM1040 has been found necessary for the survival of this organism. Most interestingly, the bacteria is able to demethylate the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) to methylmercaptopropionic acid (MMPA). DMSP is the major source of organic sulphur in the world’s oceans, forming a major part of the global sulphur cycle.<br />
<br />
A putative name 'arylformamidase' was assigned to the structure 2pbl based upon its closest mammalian homologue of the same name. Why a name based upon a mammalian homologue for a protein from such a distantly-related species remains unexplained'''(???)'''. Arylformamidase in mammals '''(?)''' is a protein involved in the tryptophan degradation pathway. Whether this name accurately reflects the protein's function in bacteria '''(?)''' needs to be assessed.<br />
<br />
<br />
FUNCTION: Catalyzes the hydrolysis of N-formyl-L-kynurenine to L-kynurenine, the second step in the conversion of tryptophan to nicotinic acid, NAD(H) and NADP(H). Required for elimination of toxic metabolites (By similarity). <br />
CATALYTIC ACTIVITY: N-formyl-L-kynurenine + H2O = formate + L-kynurenine. <br />
PATHWAY: Amino-acid degradation; L-tryptophan degradation via kynurenine pathway; L-kynurenine from L-tryptophan: step 2/2. <br />
SUBCELLULAR LOCATION: Cytoplasm, cytosol (By similarity). Nucleus (By similarity). Note=Predominantly cytosolic. Some fraction is nuclear (By similarity). <br />
SIMILARITY: Belongs to the AB hydrolase superfamily. <br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Discussion&diff=9597Arylformamidase Discussion2008-06-09T00:52:07Z<p>Basma: </p>
<hr />
<div>probable function<br />
<br />
Catalytic triad found in the canonical positions observed in a wide variety of α/β hydrolases.<br />
<br />
Functional inference from sequence similarity was restricted due to lack of supporting literature for the highest-scoring sequences. Although the function of the arylformamidase from Mus musculus has been relatively well-characterised, its structure has not been determined. Whilst a high-degree of conservation of the catalytic triad has been observed, there are structurally more-similarly proteins with an even higher degree of conservation. <br />
<br />
The carboxylesterase 2c7b was chosen for further analysis due to its high structural similarity to 2pbl. 2c7b is a thermostable carboxylesterase from an uncultured archaeon which was isolated in a thermal environmental sample (Byun 2007). This carboxylesterase belongs to a family of bacterial hormone-sensitive lipases/esterases (HSLs) known for their amino-acid sequence similarity to the mammalian hormone-sensitive lipase (ARPIGNY 1999). There were a number of other bacterial HSL proteins found to have a similar structure to 2pbl. Although in all cases there was a relatively low sequence similarity with 2pbl, regions characteristic to HSL proteins were found to be conserved indicating that 2pbl may indeed be another bacterial HSL (Arpigny 1999, see figure…). <br />
<br />
The HSL family encompasses a broad range of proteins with diverse functionality. Even though a number of HSL proteins have been determined and were included in the structure search, 2c7b was still returned as the most similar structure, indicating that 2pbl may in fact be a carboxylesterase. Specifically, carboxylesterases are involved in a broad range of reactions. Fundamentally, they catalyze the hydration of carboxyl esters into an alcohol and a carboxylic acid (see …). <br />
<br />
[[Image:carboxylesterase_reaction.png|centre|framed|'''Figure ...:''' ''Fundamental reaction mechanism of carboxylesterases.'']]<br />
<br />
Further analysis revealed that 2pbl shared greater structural similarity with thermostable esterases of the HSL family compared to its other members. As discussed, thermostable proteins such as 2c7b often exist in dimers, a possiblity which remains uninvestigated for 2pbl.<br />
<br />
<br />
== Phylogeny ==<br />
<br />
<br />
The multiple sequence alignment revealed several conserved regions accross all species, thereby indicating a high level of conservation from Bacteria through Eukaryota. Most significantly, the catalytic triad of 137S, 215E/D and 242H and many associated residues which occur in the same structural area of the protein are conserved accross all species of prokaryotes and eukaryotes. This may therefore be indicative of the conservation of functional group of residues within the protein. These included vertebrates, invertebrates, yeasts, moulds and single-celled eukaryotes. The catalytic triad is thought to be involved in thioesterase/carboxylesterase activity though the function of the protein may show variation between species.<br />
<br />
Given that the phylogeny of our protein is largely consistent with traditional taxonomic groupings of organisms and that we can find no evidence of horizontal gene transfer, the delineations between prokariotic and eukaryotic species allow us to infer that the dominant mode of inheritance is clonal from bacteria to plantae and animalia.<br />
<br />
<br />
biological implications/applications...<br />
<br />
Potential thermostability - applicability in industry.<br />
<br />
further research...<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=9595Arylformamidase Results2008-06-09T00:48:09Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''Arylformamidase exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data of such interactions.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Structural similarity'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystallization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the Archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. They are Asp 53, His 69, Gly 70, Gly 71, ''Tyr 72'', Trp 73, Gly 134, '''Ser 136''', Ala 137, Gly 138, ''His 140'', ''Ser 166'', ''Leu 168, Leu 171, Leu 174, '''Glu 214''''', '''His 241''', ''Val 244 and Leu 248'' The blue region in the below structure shows how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
The distance between the catalytic triads can be seen in figure 11. Each of the residues are liked to a turn region. This catalytic triad as stated before is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). More so the catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=9590Arylformamidase Results2008-06-09T00:39:22Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''Arylformamidase exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data of such interactions.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shows how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
The distance between the catalytic triads can be seen in figure 11. Each of the residues are liked to a turn region. This catalytic triad as stated before is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). More so the catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=9589Arylformamidase Results2008-06-09T00:38:31Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''Arylformamidase exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shows how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
The distance between the catalytic triads can be seen in figure 11. Each of the residues are liked to a turn region. This catalytic triad as stated before is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). More so the catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=9587Arylformamidase Results2008-06-09T00:37:54Z<p>Basma: </p>
<hr />
<div>== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''Arylformamidase exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shows how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
The distance between the catalytic triads can be seen in figure 11. Each of the residues are liked to a turn region. This catalytic triad as stated before is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). More so the catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Methods&diff=9584Arylformamidase Methods2008-06-09T00:35:55Z<p>Basma: </p>
<hr />
<div>'''Literature search'''<br />
<br />
A literature search was performed using the putative annotation ‘arylformamidase’. A paper by Pabarcus et al. 2007 was returned which, ironically, described the arylformamidase from Mus Musculus. <br />
<br />
'''Conservation of Catalytic Triad'''<br />
<br />
An alignment of 2pbl and the protein of interest was performed using ClustalX. Default parameters were used. Residues of the catalytic triad were identified from the paper describing it and located in the sequence. Conservation of the residue and the surrounding sequence was observed. Note: in analysing conservation of the 2c7b catalytic triad with 2pbl, the clustalW alignment was found to differ from the alignment provided as part of the DALI results.<br />
<br />
'''Sequence & Homology'''<br />
<br />
<br />
Using the query sequence [[Arylformamidase]],a BLASTP search was performed on the bacterial protein sequence using a non-redundant database. The top scoring matches to an E-value of 3e-054, 35 sequences in total, were selected. Eukaryotic homologous sequences were found using [http://www.ncbi.nlm.nih.gov/sites/entrez?itool=protein_brief&DbFrom=protein&Cmd=Link&LinkName=protein_homologene&IdsFromResult=58330909 NCBI HomoloGene]. These were appended to the list and a multiple sequence alignment was performed using CLUSTAL X. <br />
<br />
The data output from the multiple sequence alignment was bootstrapped 1000 times and a phylogenetic tree was created using the neighbour-joining algorithm. The program FigTree was used to create the visual representation of this tree.<br />
<br />
A similar BLASTP search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.<br />
<br />
Top scoring sequences from the results of the BLASTP search using the human homologue were appended to the original top scoring sequences of the results BLASTP search on the bacterial query sequence. <br />
<br />
As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorithm.<br />
<br />
<br />
'''Structure'''<br />
<br />
A number of bioinformatics databases were used to obtain the structure of arylformamidase. These include RCSB Protein Data Bank (PDB ID: 2PBL)[http://www.rcsb.org/pdb/home/home.do] for the quaternary protein structure and PDBsum [http://www.ebi.ac.uk/pdbsum/] for the secondary structure.The predicted interaction of arylformamidase with other proteins was determined using the STRING database [http://string.embl.de//]. The DALI database was used for the structural comparison of arylformamidase with other proteins [http://ekhidna.biocenter.helsinki.fi/dali_server/]. Pymol was used to annotate the conserved catalytic triad in arylformamidase structure.<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Methods&diff=9583Arylformamidase Methods2008-06-09T00:32:47Z<p>Basma: </p>
<hr />
<div>'''Literature search'''<br />
<br />
A literature search was performed using the putative annotation ‘arylformamidase’. A paper by Pabarcus et al. 2007 was returned which, ironically, described the arylformamidase from Mus Musculus. <br />
<br />
'''Conservation of Catalytic Triad'''<br />
<br />
An alignment of 2pbl and the protein of interest was performed using ClustalX. Default parameters were used. Residues of the catalytic triad were identified from the paper describing it and located in the sequence. Conservation of the residue and the surrounding sequence was observed. Note: in analysing conservation of the 2c7b catalytic triad with 2pbl, the clustalW alignment was found to differ from the alignment provided as part of the DALI results.<br />
<br />
'''Sequence & Homology'''<br />
<br />
<br />
Using the query sequence [[Arylformamidase]],a BLASTP search was performed on the bacterial protein sequence using a non-redundant database. The top scoring matches to an E-value of 3e-054, 35 sequences in total, were selected. Eukaryotic homologous sequences were found using [http://www.ncbi.nlm.nih.gov/sites/entrez?itool=protein_brief&DbFrom=protein&Cmd=Link&LinkName=protein_homologene&IdsFromResult=58330909 NCBI HomoloGene]. These were appended to the list and a multple sequece alignment was performed using CLUSTAL X. <br />
<br />
The data output from the multiple sequence alignment was bootstrapped 1000 times and a phylogenetic tree was created using the neighbour-joining algorythm. The program FigTree was used to create the visual representation of this tree.<br />
<br />
A similar BLASTP search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.<br />
<br />
Top scoring sequences from the results of the BLASTP search using the human homologue were appended to the original top scoring sequences of the results BLASTP search on the bacterial query sequence. <br />
<br />
As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorithm.<br />
<br />
<br />
'''Structure'''<br />
<br />
A number of bioinformatics databases were used to obtain the structure of arylformamidase. These include RCSB Protein Data Bank (PDB ID: 2PBL)[http://www.rcsb.org/pdb/home/home.do] for the quaternary protein structure and PDBsum [http://www.ebi.ac.uk/pdbsum/] for the secondary structure.The predicted interaction of arylformamidase with other proteins was determined using the STRING database [http://string.embl.de//]. The DALI database was used for the structural comparison of arylformamidase with other proteins [http://ekhidna.biocenter.helsinki.fi/dali_server/]. Pymol was used to make structure of arylformamidase showing the conserved catalytic triad.<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8918Arylformamidase Results2008-06-06T04:44:06Z<p>Basma: /* Structure */</p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shows how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
The distance between the catalytic triads can be seen in figure 11. Each of the residues are liked to a turn region. This catalytic triad as stated before is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). More so the catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand (see figure 12).<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|framed|centre|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8917Arylformamidase Results2008-06-06T04:36:23Z<p>Basma: /* Structure */</p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure 2) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure 2:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure 3:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure 4:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure 5:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure 6:''' ''Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure 7:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure 8:''' ''Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.'']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure 9:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shpws how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure 10:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.Image generated using Pymol'']] <br />
<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure 11:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure 12:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8916Arylformamidase Results2008-06-06T04:29:57Z<p>Basma: /* Structure */</p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
[[Image:Carboxylase.txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
A number of carboxylesterases perform their hydrolysis function using specific catalytic residues. The clustal alignment showed the conservation of the archeal carboxylesterases catalytic triad in arylformamidase. The residues are Serine (Ser) 137, Glutamate (Glu) 215 and Histidine (His) 242. Residues Ser and His were fully conserved whereas E was semi-conserved. Using the human arylformamidase it was observed that Aspartate (asp) was used for eukaryotes instead of Glu, which is used for prokaryotes.<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
Other conserved/semi-conserved residues were annotated on the structure of arylformamidase. The blue region in the below structure shpws how they all are around the catalytic triad and the unknown ligand. This clearly shows their importance for the function of the protein as they have resisted mutations.<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8907Arylformamidase Results2008-06-06T04:10:53Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
<br />
[[Image:Untitled2.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. Unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' Actual residue numbers are n+1. Image generated using Pymol'' '']]<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
<br />
<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8906Arylformamidase Results2008-06-06T04:05:06Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8905Arylformamidase Results2008-06-06T04:02:34Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the order of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
<br />
'''The catalytic triad structure'''<br />
<br />
<br />
<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8903Arylformamidase Results2008-06-06T03:59:44Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
'''Similar strucutures'''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first significant hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
The second significant hit was the carboxylesterase of Archaeoglobus fulgidus. The ligand is present in this figure.<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
<br />
'''Secondary structure analysis'''<br />
<br />
<br />
Analysis of arylformamidase's secondary structure with the Archeal carboxylsesterases showed the conservation of the pattern of occurrence of different conformational types. For instance in all three proteins, the first conformation type is a helix and then three beta strands followed by a helix and so on.<br />
<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8899Arylformamidase Results2008-06-06T03:40:28Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
'''Arylformamdiase biological structure'''<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
'''Arylformaidase interactions'''<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (i.e. absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8898Arylformamidase Results2008-06-06T03:37:22Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and experimental/biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that arylformamidase might also be a carboxylesterase.<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
The first hit from DALI was a metagenomic Archea carboxylesterase. The structure of carboxylesterase shows absence of ligands.<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY).Note: Chain B not shown. From PDB ProteinWorkshop 1.5'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B '''PDB''' ]<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8895Arylformamidase Results2008-06-06T03:27:37Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and Experimental/Biochemical data.'']]<br />
<br />
The prokaryotic arylformamidase showed no significant interaction with any of the proteins listed below (score ~0.5)<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY)'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8894Arylformamidase Results2008-06-06T03:17:36Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins/genes to occur repeatedly in close neigbourhood in the genome is not significant. So is their co-occurence (absence of linked orthologous groups across species) and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and Experimental/Biochemical data.'']]<br />
<br />
<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY)'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8893Arylformamidase Results2008-06-06T03:09:15Z<p>Basma: </p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
<br />
STRING curated database showed that human arylformamidase significantly interacted with a number of proteins. However incidence of any of those proteins to be around arylformamidase as 'neighbours' in the genome is not significant. So is their co-occurence and co-expression across the genome.<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins from curated STRING database (significant score). There is no significant evidence for Neighborhood in the genome, Gene fusions, Cooccurence across genomes, Co-Expression and Experimental/Biochemical data.'']]<br />
<br />
<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY)'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_References&diff=8892Arylformamidase References2008-06-06T02:53:22Z<p>Basma: </p>
<hr />
<div> ''Pabarcus MK, Casida JE.''<br />
'''[http://www.ncbi.nlm.nih.gov/pubmed/15935693?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Cloning, expression, and catalytic triad of recombinant arylformamidase.]'''<br />
Protein Expr Purif. Nov;44(1):39-44.<br />
<br />
''De Simone G, Menchise V, Manco G, Mandrich L, Sorrentino N, Lang D, Rossi M, Pedone C.''<br />
'''[http://www.ncbi.nlm.nih.gov/pubmed/11846563?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum The crystal structure of a hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus.]'''<br />
J Mol Biol. 2001 Nov 30;314(3):507-18.<br />
<br />
''Byun JS, Rhee JK, Kim ND, Yoon J, Kim DU, Koh E, Oh JW, Cho HS.''<br />
'''[http://www.ncbi.nlm.nih.gov/pubmed/17625021?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties.]'''<br />
BMC Struct Biol. 2007 Jul 12;7:47.<br />
<br />
2-''Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype''<br />
http://www.ncbi.nlm.nih.gov/pubmed/12007602?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DiscoveryPanel.Pubmed_Discovery_RA&linkpos=2&log$=relatedarticles&dbfrom=pubmed<br />
<br />
<br />
[http://www.ncbi.nlm.nih.gov/blast/Blast.cgi NCBI: BLAST]<br />
<br />
[http://www.ncbi.nlm.nih.gov/sites/entrez?itool=protein_brief&DbFrom=protein&Cmd=Link&LinkName=protein_homologene&IdsFromResult=58330909 NCBI HomoloGene: Arylformamidase]<br />
<br />
[http://www.clustal.org/ CLUSTAL Homepage]<br />
<br />
[http://tree.bio.ed.ac.uk/software/figtree/ Fig Tree]<br />
<br />
[http://microbewiki.kenyon.edu/index.php/Silicibacter_pomeroyi MicrobeWiki: Silicibacter pomeroyi]<br />
<br />
[http://www.rcsb.org/pdb/home/home.do/ RCSB PDB] <br />
<br />
[http://www.ebi.ac.uk/pdbsum/ PDBSUM]<br />
<br />
[http://string.embl.de// STRING]<br />
<br />
[http://ekhidna.biocenter.helsinki.fi/dali_server/ DALI]<br />
<br />
<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Methods&diff=8891Arylformamidase Methods2008-06-06T02:50:21Z<p>Basma: </p>
<hr />
<div>'''Literature search'''<br />
<br />
A literature search was performed using the putative annotation ‘arylformamidase’. A paper by Pabarcus et al. 2007 was returned which, ironically, described the arylformamidase from Mus Musculus. <br />
<br />
'''Conservation of Catalytic Triad'''<br />
<br />
An alignment of 2pbl and the protein of interest was performed using ClustalX. Default parameters were used. Residues of the catalytic triad were identified from the paper describing it and located in the sequence. Conservation of the residue and the surrounding sequence was observed. Note: in analysing conservation of the 2c7b catalytic triad with 2pbl, the clustalW alignment was found to differ from the alignment provided as part of the DALI results.<br />
<br />
'''Sequence & Homology'''<br />
<br />
<br />
Using the query sequence [[Arylformamidase]],a BLASTP search was performed on the bacterial protein sequence using a non-redundant database. The top scoring matches to an E-value of 3e-054, 35 sequences in total, were selected. Eukaryotic homologous sequences sequences were found using [http://www.ncbi.nlm.nih.gov/sites/entrez?itool=protein_brief&DbFrom=protein&Cmd=Link&LinkName=protein_homologene&IdsFromResult=58330909 NCBI HomoloGene]. These were appended to the list and a multple sequece alignment was performed using CLUSTAL X. <br />
<br />
The data output from the multiple sequence alignment was bootstrapped 1000 times and a phylogenetic tree was created using the neighbour-joining algorythm. The program FigTree was used to create the visual representation of this tree.<br />
<br />
A similar BLASTP search was performed using the human homologue to our query sequence. 126 of the top scoring matches were selected for a multiple sequence alignment. This was the minimum number of sequences which would also include the query sequence. The sequences were aligned, bootstrapped and a tree created as above. The tree revealed some questionable matches, joining humans with pufferfish for instance, which, whilst evolutionarily interesting poses more questions than answers.<br />
<br />
Top scoring sequences from the results of the BLASTP search using the human homologue were appended to the original top scoring sequences of the results BLASTP search on the bacterial query sequence. <br />
<br />
As above, using CLUSTAL X, a multiple sequence alignment was generated, the data was then bootstrapped 1000 times and a phylogenetic tree generated using the neighbour-joining algorithm.<br />
<br />
<br />
'''Structure'''<br />
<br />
A number of bioinformatics databases were used to obtain the structure of arylformamidase. These include RCSB Protein Data Bank (PDB ID: 2PBL)[http://www.rcsb.org/pdb/home/home.do] for the quaternary protein structure and PDBsum [http://www.ebi.ac.uk/pdbsum/] for the secondary structure.The predicted interaction of arylformamidase with other proteins was determined using the STRING database [http://string.embl.de//]. The DALI database was used for the structural comparison of arylformamidase with other proteins [http://ekhidna.biocenter.helsinki.fi/dali_server/]. Pymol was used to make structure of arylformamidase showing the conserved catalytic triad.<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Introduction&diff=8890Arylformamidase Introduction2008-06-06T02:39:22Z<p>Basma: </p>
<hr />
<div>why it was chosen for structural determination<br />
<br />
The structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG) (see figure 2). The organism is ''Silicibacter SP. TM1040'' and the protein expression system is ''Escherichia Coli'' using a plasmid as the vector. A resolution of 1.79 A was achieved with an R-value of 0.224 and an R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure. <br />
<br />
[[Image:Arylformamidase the whole protein.PNG|centre|framed|'''Figure 2:''' ''An overview of 2pbl exhibiting all chains. The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The red molecule in the chain structure is an unknown ligand. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). ''Image generated using PDB ProteinWorkshop 1.5.'']] <br />
<br />
Upon crystallisation, 2pbl formed a tetramer structure. However, structures formed upon crystalisation do not always denote the functional form of a protein which can exist as a dimer or oligomer as well. In fact, such forms may have been evolutionary selected for to confer features such as thermostability (Byun 2007). By examining the crystal structure and … , we have attempted to deduce the functional form of 2pbl.<br />
<br />
'Arylformamidase' heralds from ''Silibacter sp. TM1040'', a member of the ''Roseobacter'' clade of alpha-proteobacteria. It was first isolated as part of an investigation into the role of bacteria in the physiology and toxigenesis of the dinoflagellate Pfiestera piscicida. Silicibacter sp. TM1040 has been found necessary for the survival of this organism. Most interestingly, the bacteria is able to demethylate the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) to methylmercaptopropionic acid (MMPA). DMSP is the major source of organic sulphur in the world’s oceans, forming a major part of the global sulphur cycle. <br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8889Arylformamidase Results2008-06-06T02:35:11Z<p>Basma: /* Structure */</p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure) even though the "whole" protein is shown to be interacting with chains A, B, C and D. The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins'']]<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY)'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8888Arylformamidase Results2008-06-06T02:33:18Z<p>Basma: /* Structure */</p>
<hr />
<div>== Contents ==<br />
<br />
== Structure ==<br />
<br />
The functional biological structure of arylformamidase is assumed by PDB to be a monomer (see figure). The unknown ligand is shown in red and is composed of nine oxygen molecules.<br />
<br />
[[Image:ChainA1.PNG|centre|framed|'''Figure:''' ''2pbl exhibiting solely chain A. The unknown ligand (red) contains a ring composed of 9 oxygen molecules. The green sphere is a chloride ion. The protein backbone is coloured by conformation type: turn (blue), coil (pink), helix (green) and strand (purple). Image generated using PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Confidence_interaction_with_names.png|centre|framed|'''Figure ?:''' ''Interaction of human arylformamidase (AFMID) with other proteins'']]<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
[[Image:Examplec.jpg|centre|framed|'''Figure ?:''' ''Interaction of 2pbl from Silicibacter Sp. with other proteins'']]<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
[[Image:ChainA 2c7b.PNG|centre|framed|'''Figure ?:''' ''Metagenomic Archea Carboxylesterase (Chain A ONLY)'']] <br />
<br />
[[Image:Carboxylase.txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
[[Image:ChainA 1jji.PNG|centre|framed|'''Figure: Archaeoglobus fulgidus Carboxylesterase exhibiting chain A only. From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
[http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI '''PDB''']<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
[[Image:PDBSum pblA.PNG|centre|framed|'''Figure ?:''' ''PDBSum output for arylformamidase. Image courtesy of PDBSUM.'']] <br />
<br />
[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html PDBSUM]<br />
<br />
[[Image:Pdbsums archeal.PNG|centre|framed|'''Figure: Archeon Carboxylesterase secondary structure. Image courtesy of PDBSUM.''']] <br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:Cat triad red.PNG|centre|framed|'''Figure:''' ''The conserved residues of arylformamidase. The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.'']] <br />
<br />
''Image generated using Pymol'' <br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG|framed|centre|'''Figure ?:''' ''The catalytic triad. The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI). From PDB ProteinWorkshop 1.5'']]<br />
<br />
[[Image:Cat triad 1jji.PNG|centre|framed|'''Figure ?:''' ''The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'']]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
== Function == <br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
== Sequence & Homology ==<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
[[Image:NewBOOT1000tree.png|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).'']]<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
[[Image:BacterANDhomoTREE.jpg|centre|framed|'''Figure ...:''' ''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).'']]<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Structure&diff=8492Arylformamidase Structure2008-06-03T10:10:23Z<p>Basma: </p>
<hr />
<div>=='''Methods'''==<br />
<br />
The structure of arylfromamidase was obtained from RCSB Protein Data Bank (PDB ID: 2PBL).<br />
http://www.rcsb.org/pdb/home/home.do<br />
<br />
The predicted interaction arylformamidase with other proteins was determined using the STRING database (STRING: Search Tool for the Retrieval of Interacting Genes/Proteins). http://string.embl.de// <br />
<br />
The DALI database was used for the structural comparison of arylformamidase with other proteins. http://ekhidna.biocenter.helsinki.fi/dali_server/<br />
<br />
PDBsum database was used to determine the secondary structure of arylformamidase.<br />
http://www.ebi.ac.uk/pdbsum/<br />
<br />
<br />
<br />
== '''Results''' ==<br />
<br />
<br />
== Structure of Arylformamidase ==<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
<br />
<br />
<br />
'''Figure: Arylformamidase (All Chains)'''<br />
<br />
<br />
[[Image:Arylformamidase the whole protein.PNG]] <br />
<br />
<br />
''The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure:''Chain A'' of arylformamidase'''<br />
<br />
<br />
[[Image:ChainA1.PNG]] <br />
<br />
<br />
''The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5'' <br />
<br />
'''''The protein backbone is coloured by conformation type:'''''<br />
<br />
'''''Turn - blue'''''<br />
<br />
'''''Coil- pink'''''<br />
<br />
'''''Helix- green'''''<br />
<br />
'''''strand- purple'''''<br />
<br />
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Confidence_interaction_with_names.png]]<br />
<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
== '''Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Examplec.jpg]]<br />
<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
== '''DALI OUTPUT''' ==<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
<br />
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 2c7b.PNG]] <br />
<br />
[[Image:Carboxylase.txt ]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 1jji.PNG]] <br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]<br />
<br />
''Note: Chains B, C & D not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
== '''Secondary structure analysis''' ==<br />
<br />
<br />
<br />
'''PDBSum output for arylformamidase'''<br />
<br />
[[Image:PDBSum pblA.PNG]] <br />
<br />
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]<br />
<br />
<br />
'''Figure: Archeon Carboxylesterase secondary structure'''<br />
<br />
[[Image:Pdbsums archeal.PNG]] <br />
<br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
== '''The conservation of the ser/his/asp catalytic triad''' ==<br />
<br />
[[Image:Catalytic triad conversation.PNG]]<br />
<br />
''Yellow indicates conservation''<br />
<br />
''Blue indicates semi-conservation''<br />
<br />
<br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:Untitled2.PNG]]<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The conserved residues of arylformamidase'''<br />
<br />
<br />
[[Image:Cat triad red.PNG]] <br />
<br />
<br />
''The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG]]<br />
<br />
''The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)''<br />
<br />
'' From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''<br />
<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG]]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase&diff=8489Arylformamidase2008-06-03T10:07:41Z<p>Basma: </p>
<hr />
<div>[[Image:Pretty protein.PNG|centre|framed|'''Figure 1.''' ''Arylformamidase'']]<br />
<br />
''by Basma Al Alaiwat, Sebastian Mynott and Thomas Parker''<br />
<br />
== Abstract ==<br />
<br />
2pbl, initially annotated an arylformamidase, was isolated from Silibacter sp. TM1040 and its structure determined by the JCSG.<br />
Based on this structure, and using structural, functional and evolutionary analysis, we have further characterised 2pbl. It was found to contain a conserved functional region that is present in a broad range of proteins in many taxonomic groups. Specifically, it was found to share similar structural and sequence characteristics of the HSL family of esterases. Although the specific function is still not known, 2pbl may present novel industrial applications.<br />
<br />
<br />
<br />
<br />
<br />
== Contents ==<br />
<br />
[[Arylformamidase Introduction|Introduction]] <br />
<br />
[[Arylformamidase Results|Results]] <br />
<br />
[[Arylformamidase Discussion|Discussion]]<br />
<br />
[[Arylformamidase Methods|Methods]] <br />
<br />
[[Arylformamidase Additional Materials|Additional Materials]]<br />
<br />
[[Arylformamidase References|References]]<br />
<br />
== Presentations ==<br />
<br />
[[Arylformamidase Sequence & Homology | Sequence & Homology]] - ''Sebastian Mynott''<br />
<br />
[[Arylformamidase Structure | Structure]] - ''Basma Al Alaiwat''<br />
<br />
[[Arylformamidase Function | Function]] - ''Thomas Parker''</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase&diff=8487Arylformamidase2008-06-03T10:06:27Z<p>Basma: /* Abstract */</p>
<hr />
<div>''by Basma Al Alaiwat, Sebastian Mynott and Thomas Parker''<br />
<br />
== Abstract ==<br />
<br />
2pbl, initially annotated an arylformamidase, was isolated from Silibacter sp. TM1040 and its structure determined by the JCSG.<br />
Based on this structure, and using structural, functional and evolutionary analysis, we have further characterised 2pbl. It was found to contain a conserved functional region that is present in a broad range of proteins in many taxonomic groups. Specifically, it was found to share similar structural and sequence characteristics of the HSL family of esterases. Although the specific function is still not known, 2pbl may present novel industrial applications.<br />
<br />
<br />
<br />
<br />
[[Image:Pretty protein.PNG|centre|framed'''Figure 1.''' ''Arylformamidase'']]<br />
<br />
== Contents ==<br />
<br />
[[Arylformamidase Introduction|Introduction]] <br />
<br />
[[Arylformamidase Results|Results]] <br />
<br />
[[Arylformamidase Discussion|Discussion]]<br />
<br />
[[Arylformamidase Methods|Methods]] <br />
<br />
[[Arylformamidase Additional Materials|Additional Materials]]<br />
<br />
[[Arylformamidase References|References]]<br />
<br />
== Presentations ==<br />
<br />
[[Arylformamidase Sequence & Homology | Sequence & Homology]] - ''Sebastian Mynott''<br />
<br />
[[Arylformamidase Structure | Structure]] - ''Basma Al Alaiwat''<br />
<br />
[[Arylformamidase Function | Function]] - ''Thomas Parker''</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Structure&diff=8486Arylformamidase Structure2008-06-03T10:05:03Z<p>Basma: </p>
<hr />
<div>=='''Methods'''==<br />
<br />
The structure of arylfromamidase was obtained from RCSB Protein Data Bank (PDB ID: 2PBL).<br />
http://www.rcsb.org/pdb/home/home.do<br />
<br />
The predicted interaction arylformamidase with other proteins was determined using the STRING database (STRING: Search Tool for the Retrieval of Interacting Genes/Proteins). http://string.embl.de// <br />
<br />
The DALI database was used for the structural comparison of arylformamidase with other proteins. http://ekhidna.biocenter.helsinki.fi/dali_server/<br />
<br />
PDBsum database was used to determine the secondary structure of arylformamidase.<br />
http://www.ebi.ac.uk/pdbsum/<br />
<br />
<br />
<br />
== '''Results''' ==<br />
<br />
<br />
<br />
<br />
<br />
[[Image:Pretty protein.PNG]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
== Structure of Arylformamidase ==<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
<br />
<br />
<br />
'''Figure: Arylformamidase (All Chains)'''<br />
<br />
<br />
[[Image:Arylformamidase the whole protein.PNG]] <br />
<br />
<br />
''The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure:''Chain A'' of arylformamidase'''<br />
<br />
<br />
[[Image:ChainA1.PNG]] <br />
<br />
<br />
''The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5'' <br />
<br />
'''''The protein backbone is coloured by conformation type:'''''<br />
<br />
'''''Turn - blue'''''<br />
<br />
'''''Coil- pink'''''<br />
<br />
'''''Helix- green'''''<br />
<br />
'''''strand- purple'''''<br />
<br />
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Confidence_interaction_with_names.png]]<br />
<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
== '''Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Examplec.jpg]]<br />
<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
== '''DALI OUTPUT''' ==<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
<br />
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 2c7b.PNG]] <br />
<br />
[[Image:Carboxylase.txt ]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 1jji.PNG]] <br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]<br />
<br />
''Note: Chains B, C & D not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
== '''Secondary structure analysis''' ==<br />
<br />
<br />
<br />
'''PDBSum output for arylformamidase'''<br />
<br />
[[Image:PDBSum pblA.PNG]] <br />
<br />
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]<br />
<br />
<br />
'''Figure: Archeon Carboxylesterase secondary structure'''<br />
<br />
[[Image:Pdbsums archeal.PNG]] <br />
<br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
== '''The conservation of the ser/his/asp catalytic triad''' ==<br />
<br />
[[Image:Catalytic triad conversation.PNG]]<br />
<br />
''Yellow indicates conservation''<br />
<br />
''Blue indicates semi-conservation''<br />
<br />
<br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:Untitled2.PNG]]<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The conserved residues of arylformamidase'''<br />
<br />
<br />
[[Image:Cat triad red.PNG]] <br />
<br />
<br />
''The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG]]<br />
<br />
''The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)''<br />
<br />
'' From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''<br />
<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG]]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=File:Pretty_protein.PNG&diff=8485File:Pretty protein.PNG2008-06-03T10:04:00Z<p>Basma: </p>
<hr />
<div></div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Results&diff=8466Arylformamidase Results2008-06-03T09:28:45Z<p>Basma: </p>
<hr />
<div>most similar sequence - catalytic triad, structure with highlighted<br />
<br />
The most similar sequence with functional information available was that of an arylformamidase isolated from the liver of Mus musculus (see figure ...). A functional analysis of this protein has been performed identifying a catalytic triad using site-directed mutagenesis (Pabarcus et al. 2007). Conservation of this catalytic triad with 2pbl was assessed. Both residues S162 and H279 were found to be conserved in relatively conserved regions of the alignment. However, D247 had undergone a semi-conservative substitution. These residues correlated to S136, E214 and H241 of 2pbl which were subsequently located on the tertiary structure and determined to be sufficiently proximal to one another for catalysis (see figure...).<br />
<br />
[[Image:arylformamidase_alignment.png|centre|framed|'''Figure 3:''' ''Conservation of the catalytic triad between Arylformamidase and 2pbl.'']]<br />
<br />
most similar structure - catalytic triad, structure with highlighted<br />
<br />
2pbl was found to share most structural similarity with a thermostable carboxylesterase from an uncultured archaeon (PDB ID: 2c7b; see figure ...). 2c7b shares a 16% sequence identity with 2pbl. From its structure, a catalytic triad has been identified (how?). To substantiate any functional similarity between 2pbl and 2c7b, conservation of the 2c7b catalytic triad was analysed (see figure ...). All three residues were found to be conserved, though H... and E... were found to match is less conserved regions.<br />
<br />
[[Image:2c7b_alignment.png|centre|framed|'''Figure 1:''' ''Conservation of the catalytic triad between 2cb7 and 2pbl.'']]<br />
<br />
[[Image:HSL_alignment.png|centre|framed|'''Figure 2:''' ''Alignment with other HSL family members.'']]<br />
<br />
<br />
== Sequence & Homology ==<br />
<br />
<br />
Figure 1 shows that the query sequence "Arylformamidase" grouped with bacterial sequences, shown cloured in Blue. The bootstrap values reveal low confidence with many of the nodes occurring lower down on the phylogenetic tree revealing a possible explanation for certain closely related species to be grouped into separate clades. However, despite low bootstrap scores, the grouping does reliably separate prokaryotes from eukaryotes and the eukaryotes themsselves are clearly distinguished between yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).<br />
<br />
<br />
'''Figure 1.'''<br />
<br />
[[Image:NewBOOT1000tree.png]]<br />
<br />
''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequnces using a non-redundant database and homologous eukaryotic sequences sourced from NCBI HomoloGene. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotic organisms shown in Blue, from eukaryote yeasts and moulds (shown in Green), plants (Dark Green), invertebrates (Orange) and vertebrates (shown in Red).''<br />
<br />
<br />
To further elucidate the phylogeny of the Arylformamidase protein, top scoring matches of bacterial homologues were appended with top scoring matches of eukaryotic homologues. Figure 2 is largely consistent with traditional taxonomic groupings of organisms. Specifically, it reveals greater statistical confidence in the separation of prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).<br />
<br />
<br />
'''Figure 2.'''<br />
<br />
[[Image:BacterANDhomoTREE.jpg]]<br />
<br />
''Unrooted phylogenetic tree of highest scoring results from a BLASTP search of bacterial sequences and highest scoring results of a BLASTP search on a homologous human sequence. Branch lengths are related to phylogenetic distance and node numbers refer to Bootstrap values. On this tree "Arylformamidase" refers to the Silicibacter species from which our sequence originated. The colour coding distinguishes prokaryotes (Blue and Green) and eukaryotes (invertebrates are shown in Orange; vertebrates are in Red).''<br />
<br />
In general, members of the same genus have been grouped together on these phylogenetic trees with some notable exceptions. For instance, Silicibacter, the species from which we derived our protein, occurs on disparate branches of the tree.<br />
<br />
<br />
<br />
== Structure of Arylformamidase ==<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
<br />
<br />
<br />
'''Figure: Arylformamidase (All Chains)'''<br />
<br />
<br />
[[Image:Arylformamidase the whole protein.PNG]] <br />
<br />
<br />
''The image above shows the chains A (upper right), B (upper left), C (lower right) & D (lower left) interacting. The molecules in the middle of chains A & B and chains C & D is phosphate ion (PO4). The green molecule between chain B & D is a magnesium ion (Mg). These ions aren't biologically significant and could only be an artefact. When crystallizing proteins they often form complexes (dimer, tetramers etc) but that doesn't mean that the functional structure is the same. They could be functional monomers. The chains in the protein of interest exist as individual functional units because in the PDB file it assumes the functional biological molecule as a monomer.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure:''Chain A'' of arylformamidase'''<br />
<br />
<br />
[[Image:ChainA1.PNG]] <br />
<br />
<br />
''The red molecule in the middle is an unknown ligand containing a ring composed of 9 oxygen molecules. The green sphere is a chloride ion.''<br />
<br />
''Image from PDB ProteinWorkshop 1.5'' <br />
<br />
'''''The protein backbone is coloured by conformation type:'''''<br />
<br />
'''''Turn - blue'''''<br />
<br />
'''''Coil- pink'''''<br />
<br />
'''''Helix- green'''''<br />
<br />
'''''strand- purple'''''<br />
<br />
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Confidence_interaction_with_names.png]]<br />
<br />
<br />
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''<br />
<br />
''1- Neighborhood in the genome''<br />
<br />
''2- Gene fusions '' <br />
<br />
''3- Cooccurence across genomes '' <br />
<br />
''4- Co-Expression '' <br />
<br />
''5- Experimental/Biochemical data''<br />
<br />
== '''Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==<br />
<br />
<br />
<br />
[[Image:Examplec.jpg]]<br />
<br />
<br />
TM1040_2226 ''Tryptophan 2,3-dioxygenase (279 aa)''<br />
<br />
TM1040_2225 ''Kynureninase (396 aa)''<br />
<br />
TM1040_2493 ''Succinic semialdehyde dehydrogenase (490 aa)''<br />
<br />
TM1040_1862 ''Hypothetical protein (212 aa)''<br />
<br />
TM1040_2491 ''Creatinase (402 aa'')<br />
<br />
TM1040_2736 ''Transketolase, putative (794 aa)''<br />
<br />
<br />
''There is no significant evidence for these interactions (score= ~0.5)''<br />
<br />
== '''DALI OUTPUT''' ==<br />
<br />
<br />
<br />
The DALI tool produces proteins that are structurally similar to the protein of interest. <br />
<br />
The search result showed similarities to mostly carboxylesterases/hydrolases. Hence there is strong evidence that our protein might also be a carboxylesterase.<br />
<br />
[[Image:DALI RESULT.txt]]<br />
<br />
<br />
<br />
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 2c7b.PNG]] <br />
<br />
[[Image:Carboxylase.txt ]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]<br />
<br />
''Note: Chain B not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''<br />
<br />
[[Image:ChainA 1jji.PNG]] <br />
<br />
[[Image:Carboxylesterase (archaeon).txt]]<br />
<br />
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]<br />
<br />
''Note: Chains B, C & D not shown''<br />
<br />
''From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
''Both of the above Archaeal carboxylesterases' chains exist as monomers (from literature). Hence it is expected that our protein exists as a monomer but during crystalization it interacts with its chains.''<br />
<br />
== '''Secondary structure analysis''' ==<br />
<br />
<br />
<br />
'''PDBSum output for arylformamidase'''<br />
<br />
[[Image:PDBSum pblA.PNG]] <br />
<br />
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]<br />
<br />
<br />
'''Figure: Archeon Carboxylesterase secondary structure'''<br />
<br />
[[Image:Pdbsums archeal.PNG]] <br />
<br />
<br />
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''<br />
<br />
''Images from PDBsum''<br />
<br />
== '''The conservation of the ser/his/asp catalytic triad''' ==<br />
<br />
[[Image:Catalytic triad conversation.PNG]]<br />
<br />
''Yellow indicates conservation''<br />
<br />
''Blue indicates semi-conservation''<br />
<br />
<br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:Untitled2.PNG]]<br />
<br />
<br />
''The above image shows the conserved residues of the catalytic triad in arylformamidase, with the unknown ligand (Blue) protruding from a surface groove. The residues are serine 136, Histidine 241 and Glutamate 214. '''Note:''' The actual residue numbers are n+1''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The conserved residues of arylformamidase'''<br />
<br />
<br />
[[Image:Cat triad red.PNG]] <br />
<br />
<br />
''The blue region shows the residues conserved among species. It is mostly around the unknown ligand. The conserved residues were obtained from observing the clustal alignment.''<br />
<br />
''Image generated using Pymol'' <br />
<br />
<br />
'''Figure: The catalytic triad'''<br />
<br />
<br />
[[Image:CATALYTIC TRIAD 1.PNG]]<br />
<br />
''The above image shows the distance between the catalytic triad conserved residues and how each amino acid is linked to a turn region. This catalytic triad is also conserved in the Metagenomic Archea Carboxylesterase (PDB ID 2C7B) and the Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)''<br />
<br />
'' From PDB ProteinWorkshop 1.5''<br />
<br />
<br />
<br />
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''<br />
<br />
<br />
<br />
[[Image:Cat triad 1jji.PNG]]<br />
<br />
<br />
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''<br />
<br />
[[Arylformamidase | Return to the main page...]]<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Introduction&diff=8465Arylformamidase Introduction2008-06-03T09:27:58Z<p>Basma: </p>
<hr />
<div>why it was chosen for structural determination<br />
<br />
how its annotation arrived at 'arylformamidase<br />
<br />
information from pdb - structure details, etc... (perhaps picture)<br />
<br />
paragraph on silicibacter sp tm1040 - evolutionary info (where it fits in) (diagram)<br />
<br />
'Arylformamidase' heralds from Silibacter sp. TM1040, a member of the Roseobacter clade of alpha-proteobacteria. It was first isolated as part of an investigation into the role of bacteria in the physiology and toxigenesis of the dinoflagellate Pfiestera piscicida. Silicibacter sp. TM1040 has been found necessary for the survival of this organism. Most interestingly, the bacteria is able to demethylate the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) to methylmercaptopropionic acid (MMPA). DMSP is the major source of organic sulphur in the world’s oceans, forming a major part of the global sulphur cycle. <br />
<br />
Upon crystallisation, 2pbl formed a tetramer structure. However, structures formed upon crystalisation do not always denote the functional form of a protein which can exist as a dimer or oligomer as well. In fact, such forms may have been evolutionary selected for to confer features such as thermostability (Byun 2007). By examining the crystal structure and … , we have attempted to deduce the functional form of 2pbl.<br />
<br />
<br />
Structure was determined using X-ray diffraction by the Joint Center for Structural Genomics (JCSG). The organism is Silicibacter SP. TM1040 and the protein expression system is Escherichia Coli (vector type: plasmid). The resolution is 1.79 A with R-value of 0.224 and R-free value of 0.270. The closer the R values are to each other, the better the quality of the structure.<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basmahttp://compbio.biosci.uq.edu.au/mediawiki/index.php?title=Arylformamidase_Methods&diff=8436Arylformamidase Methods2008-06-03T09:18:27Z<p>Basma: </p>
<hr />
<div>'''Literature search'''<br />
<br />
A literature search was performed using the putative annotation ‘arylformamidase’. A paper by Pabarcus et al. 2007 was returned which, ironically, described the arylformamidase from Mus Musculus. <br />
<br />
'''Conservation of Catalytic Triad'''<br />
<br />
An alignment of 2pbl and the protein of interest was performed using ClustalX. Default parameters were used. Residues of the catalytic triad were identified from the paper describing it and located in the sequence. Conservation of the residue and the surrounding sequence was observed. Note: in analysing conservation of the 2c7b catalytic triad with 2pbl, the clustalW alignment was found to differ from the alignment provided as part of the DALI results.<br />
<br />
'''Structure'''<br />
<br />
The structure of arylfromamidase was obtained from RCSB Protein Data Bank (PDB ID: 2PBL).<br />
http://www.rcsb.org/pdb/home/home.do<br />
<br />
The predicted interaction arylformamidase with other proteins was determined using the STRING database (STRING: Search Tool for the Retrieval of Interacting Genes/Proteins). http://string.embl.de// <br />
<br />
The DALI database was used for the structural comparison of arylformamidase with other proteins. http://ekhidna.biocenter.helsinki.fi/dali_server/<br />
<br />
PDBsum database was used to determine the secondary structure of arylformamidase.<br />
http://www.ebi.ac.uk/pdbsum/<br />
<br />
<br />
[[Arylformamidase | Return to the main page...]]</div>Basma