Arylformamidase Results: Difference between revisions

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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.
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.


== Structure of Arylformamidase ==
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.
'''Figure: Arylformamidase (All Chains)'''
[[Image:Arylformamidase the whole protein.PNG]]       
''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.''
''Image from PDB ProteinWorkshop 1.5''
'''Figure:''Chain A'' of arylformamidase'''
[[Image:ChainA1.PNG]] 
''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.''
''Image from PDB ProteinWorkshop 1.5''
'''''The protein backbone is coloured by conformation type:'''''
'''''Turn - blue'''''
'''''Coil- pink'''''
'''''Helix- green'''''
'''''strand- purple'''''
== '''Interaction of human arylformamidase (AFMID) with other proteins''' ==
[[Image:Confidence_interaction_with_names.png]]
''The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:''
''1- Neighborhood in the genome''
''2- Gene fusions ''   
''3- Cooccurence across genomes  ''
''4- Co-Expression  '' 
''5- Experimental/Biochemical data''
== '''Interaction of  Silicibacter Sp. arylformamidase (AFMID) with other proteins''' ==
[[Image:Examplec.jpg]]
TM1040_2226          ''Tryptophan 2,3-dioxygenase (279 aa)''
TM1040_2225          ''Kynureninase (396 aa)''
TM1040_2493          ''Succinic semialdehyde dehydrogenase (490 aa)''
TM1040_1862          ''Hypothetical protein (212 aa)''
TM1040_2491          ''Creatinase (402 aa'')
TM1040_2736          ''Transketolase, putative (794 aa)''
''There is no significant evidence for these interactions (score= ~0.5)''
== '''DALI OUTPUT''' ==
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 our protein might also be a carboxylesterase.
[[Image:DALI RESULT.txt]]
'''Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)'''
[[Image:ChainA 2c7b.PNG]]   
[[Image:Carboxylase.txt ]]
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=2C7B link title]
''Note: Chain B not shown''
''From PDB ProteinWorkshop 1.5''
'''Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)'''
[[Image:ChainA 1jji.PNG]] 
[[Image:Carboxylesterase (archaeon).txt]]
'''PDB''' [http://www.rcsb.org/pdb/explore/explore.do?structureId=1JJI link title]
''Note: Chains B, C & D not shown''
''From PDB ProteinWorkshop 1.5''
''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.''
== '''Secondary structure analysis''' ==
'''PDBSum output for arylformamidase'''
[[Image:PDBSum pblA.PNG]]
PDBSUM [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=2pbl&template=main.html]
'''Figure: Archeon Carboxylesterase secondary structure'''
[[Image:Pdbsums archeal.PNG]] 
''The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.''
''Images from PDBsum''
== '''The conservation of the ser/his/asp catalytic triad''' ==
[[Image:Catalytic triad conversation.PNG]]
''Yellow indicates conservation''
''Blue indicates semi-conservation''
'''Figure: The catalytic triad'''
[[Image:Untitled2.PNG]]
''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''
''Image generated using Pymol''
'''Figure: The conserved residues of arylformamidase'''
[[Image:Cat triad red.PNG]]
''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''
'''Figure: The catalytic triad'''
[[Image:CATALYTIC TRIAD 1.PNG]]
''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''
'''Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)'''
[[Image:Cat triad 1jji.PNG]]
''The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.''
[[Arylformamidase | Return to the main page...]]




[[Arylformamidase | Return to the main page...]]
[[Arylformamidase | Return to the main page...]]

Revision as of 09:28, 3 June 2008

most similar sequence - catalytic triad, structure with highlighted

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...).

Figure 3: Conservation of the catalytic triad between Arylformamidase and 2pbl.

most similar structure - catalytic triad, structure with highlighted

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.

Figure 1: Conservation of the catalytic triad between 2cb7 and 2pbl.
Figure 2: Alignment with other HSL family members.


Sequence & Homology

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).


Figure 1.

NewBOOT1000tree.png

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).


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).


Figure 2.

BacterANDhomoTREE.jpg

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).

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.


Structure of Arylformamidase

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.



Figure: Arylformamidase (All Chains)


Arylformamidase the whole protein.PNG


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.

Image from PDB ProteinWorkshop 1.5


Figure:Chain A of arylformamidase


ChainA1.PNG


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.

Image from PDB ProteinWorkshop 1.5

The protein backbone is coloured by conformation type:

Turn - blue

Coil- pink

Helix- green

strand- purple

Interaction of human arylformamidase (AFMID) with other proteins

Confidence interaction with names.png


The interaction between the proteins have been determined from curated STRING database (significant score). However there is no significant evidence for:

1- Neighborhood in the genome

2- Gene fusions

3- Cooccurence across genomes

4- Co-Expression

5- Experimental/Biochemical data

Interaction of Silicibacter Sp. arylformamidase (AFMID) with other proteins

Examplec.jpg


TM1040_2226 Tryptophan 2,3-dioxygenase (279 aa)

TM1040_2225 Kynureninase (396 aa)

TM1040_2493 Succinic semialdehyde dehydrogenase (490 aa)

TM1040_1862 Hypothetical protein (212 aa)

TM1040_2491 Creatinase (402 aa)

TM1040_2736 Transketolase, putative (794 aa)


There is no significant evidence for these interactions (score= ~0.5)

DALI OUTPUT

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 our protein might also be a carboxylesterase.

File:DALI RESULT.txt


Figure: Metagenomic Archea Carboxylesterase (Chain A ONLY)

ChainA 2c7b.PNG

File:Carboxylase.txt

PDB link title

Note: Chain B not shown

From PDB ProteinWorkshop 1.5


Figure: Archaeoglobus fulgidus Carboxylesterase (Chain A ONLY)

ChainA 1jji.PNG

File:Carboxylesterase (archaeon).txt

PDB link title

Note: Chains B, C & D not shown

From PDB ProteinWorkshop 1.5


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.

Secondary structure analysis

PDBSum output for arylformamidase

PDBSum pblA.PNG

PDBSUM [1]


Figure: Archeon Carboxylesterase secondary structure

Pdbsums archeal.PNG


The secondary structure shows the conservation of the order of different conformation types between the protein of interest and the archaeal carboxylesterases.

Images from PDBsum

The conservation of the ser/his/asp catalytic triad

Catalytic triad conversation.PNG

Yellow indicates conservation

Blue indicates semi-conservation



Figure: The catalytic triad


Untitled2.PNG


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

Image generated using Pymol


Figure: The conserved residues of arylformamidase


Cat triad red.PNG


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


Figure: The catalytic triad


CATALYTIC TRIAD 1.PNG

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


Figure: The conserved catalytic triad in Archaeoglobus fulgidus Carboxylesterase (PDB ID 1JJI)


Cat triad 1jji.PNG


The catalytic triad in Archaeoglobus fulgidus Carboxylesterase is very close to the ligand which is also present in aryformamidase.

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