3bsqA Results

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Multiple sequence alignment

Multiple sequence alignment (MSA) highlighted several residues in N-terminal region of the molecule which are highly conserved (figure 1).

MSA1.png MAS2.png MAS3.png MSA4.png MSA5.png MSA6.png MSA7.png

Figure 1: Multiple sequence alignment (MSA). Residues which are concerved across the entire sulfatase family are marked in red

Arylsulfatase K structure

Figure 2: Arylsulfatase K as a heterodimer; 3b5q chain A and B

Protein data bank profile characteris arylsulfatase as a hydrolase and a sulfatase. A Sulfatase hydrolyses sulfate esters bonds of substrates, including O-sulfates and N-sulfates as shown below.


Structural alignment

  • Three dimentional structure of arylsulfatase was aligned with other available structures using DALI server (structural alignment). Results are shown in 'figure 3'.
 No:  Chain   Z    rmsd lali nres  %id  Description
  1:  3b5q-A 73.6  0.0  464   464  100   MOLECULE: PUTATIVE SULFATASE YIDJ;                                   
  2:  3b5q-B 70.2  0.3  464   467  100   MOLECULE: PUTATIVE SULFATASE YIDJ;                                   
  3:  2qzu-A 35.1  2.5  375   465   25   MOLECULE: PUTATIVE SULFATASE YIDJ;                                   
  4:  1fsu   28.7  2.8  344   474   22   MOLECULE: N-ACETYLGALACTOSAMINE-4-SULFATASE;                         
  5:  1n2l-A 28.4  3.0  343   483   25   MOLECULE: ARYLSULFATASE A;                                           
  6:  1n2k-A 28.1  3.1  342   482   25   MOLECULE: ARYLSULFATASE A;                                           
  7:  1e2s-P 28.1  3.0  341   481   26   MOLECULE: ARYLSULFATASE A;                                           
  8:  1e3c-P 28.0  3.1  341   481   26   MOLECULE: ARYLSULFATASE A;                                           
  9:  1e33-P 28.0  3.1  342   480   25   MOLECULE: ARYLSULFATASE A;                                           
 10:  1e1z-P 27.9  3.0  341   481   26   MOLECULE: ARYLSULFATASE A;                                           
 11:  1auk   27.8  3.1  340   481   26   MOLECULE: ARYLSULFATASE A;                                           
 12:  1p49-A 27.7  2.9  338   548   24   MOLECULE: STERYL-SULFATASE;                                          
 13:  1hdh-B 27.4  3.1  365   525   23   MOLECULE: ARYLSULFATASE;                                             
 14:  1hdh-A 27.4  3.0  363   525   23   MOLECULE: ARYLSULFATASE;                                             
 15:  2rh6-A 24.3  2.6  257   382   14   MOLECULE: PHOSPHODIESTERASE-NUCLEOTIDE PYROPHOSPHATASE;              
 16:  2rh6-B 24.2  2.6  257   382   14   MOLECULE: PHOSPHODIESTERASE-NUCLEOTIDE PYROPHOSPHATASE;       

figure 3: Structural alighment of ASK. 3b5q-A and B are two chains of ASK dimer and 2qzu-A is ASK of Bacterioides fragilis. N-acetylgalactosamine-4-sulfatase, Arylsulfatase A and steryl sulfatase, also known as stroid sulfatase (STS) are most structurally similar proteins to ASK. 1hdf is the ASK of Pseudomonal auruginosa.

Arylsulfatase A has both sulfuric ester hydrolase and phosphoric monoester hydrolase activities and localised in lysosomes.
Steryl-sulfatase is a sulfuric ester hydrolase found in endoplasmin reticulums.

Arylsulfatase K interactions with other proteins

figure: 4 ASK interactions with other proteins. Gene neighbourhood evidence indicated by green lines, cooccurance evidence by dark blue lines and homology by light blue.

Subcellular interactions of arylsulfatase K were searched usnig the programme STRING, based on 'neighbourhood', 'cooccurreance' and 'homology' evidence. Results are shown below.

Input Protein

  • BT1596 Putative sulfatase yidJ (481 aa) of Bacteroides thetaiotaomicron.

Predicted Functional Partners

  • BT3796: Putative secreted sulfatase ydeN (518 aa).
  • BT1595 Transcription termination factor rho (722 aa).
  • BT1597 Two-component system sensor histidine kinase (539 aa).
  • BT3057 N-acetylgalactosamine-6-sulfatase (508 aa).
  • BT1598 Putative two-component system sensor histidine (655 aa).
  • BT3101 N-sulphoglucosamine sulphohydrolase (455 aa).
  • BT3489 Arylsulfatase B {UniProtKB/TrEMBL-Q8A219} (458 aa).

'Putative secreted sulfatase ydeN' only showed neignbourhood relationship, which means that two genes are located in close proximity. In contrast, three of other proteins showed both cooccurrence and homology evidence.

ProFunc results for ASK interacting proteins.

  • N-acetylgalactosamine-6-sulfatase cleaves the 6-sulfate groups of N-acetyl-D-galactosamine 6-sulfate units in chondroitin sulfate and D-galactose 6-sulfate units in keratan sulfate.
  • N-sulphoglucosamine sulphohydrolase is also known as heparine sulfamidase, which catalyses the hydrolysis of Sulfur-Nitrogen bonds. N-sulphoglucosamine sulphohydrolase is responsible for the degradation of glucosaminlglycan and glycan structure of extra cellular matrix.
  • N-acetylgalactosamine-4- sulfatase (Arylsulfatase B) hydrolyse the sulfate ester group from N-acetylgalactosamine 4-sulfate of dermatine sulfate. Deficiency of ASB cause a rare mucopolysaccharidosis (MPS IV; Maroteaux-Lamy syndrome)

Two sequence alignment of ASK and STS

STS was chosen to be the most similar enzyme to ASK due to the shired subcellylar localization. Two sequence alignmnt between ASK and STS is shown is figure: 5.

Figure: 5 Two sequence alignment of ASK with STS using 'SIM' server, alignment methode' BLOSUM62' with gap penalty of 5 and gap extension penalty of 2

Figure 5: Catalytic site of Arylsulfatase A (ASA) with the Magnesium ion bound.

Figure 6: Catalytic site of STS with calsium ino bound (Blue). Cation binding residues are marked in 'cyan' and catalytic residues are marked in pink

figure 7: Catalytic site of Arylsulfatase K (ASK) with conserved catalytic residues. Residues marked in grey are possible to be involved in divalent metal binding , while residues marked in magenta are highly conserved with those of Arylsulfatase A (ASA) and steroid sulfatase (STS). Histidine (H) shown in cyan is not strictly consetved in MSA, but the only available nucleophile in the close proximity

Active site and surface architecture

Figure 8: Surface of the ASK N-terminal catalytic site. It is buried deep in the moleculae, conected to the exterior via a narrow passage. This feature is also conserved with the STS catalytic site which is connected to the exterior via a substrate-entry path
Figure 9: Surface of STS and catalytic site. Substrate moving path is much narrover and longer compared to that of ASK
Figure 10: Catalytic site of ASA. Substrate entry path is wider and shorter compaired to that of STS and much similar to the catalytic site of ASK.

Possible function and likely substrates of ASK

ASA is a water soluble enzyme found in lysosomes, where the catalytic site is closer to the surface and substrate-entry path is narrower and wider in contrast to that of STS. Structural comparison shows ASK catalsytic site architecture is much similar to that of ASA, eventhough subcellular localization is very different. ASA binds to membrane lipid sulphatides and show a number of hydrophibic patches near the catalytic site. However an exact substrate binding site is not defined. ASK structure also show number of hydrophobic residues on the surface (Figure 11) indicating the hydrophobic nature of the substrate it binds.

figure 11: Surfece of ASK show number of hydrophobic residues (marked in orange). These may be involved in substrate binding

Multiple sequence alignment

  • The multiple sequence alignment constructed in clustalx shows similiarity extended across the entire sequences, especially observed in N-termainal. The N-terminal showed conserved regions of amino acids containing arginine and histidine residues, which have found to be involved in the assembly of active sites of most arylsulfatases.

Figure 12
Multiple sequence alignment (MSA) of proteins showing sequence homology to arylsulfatse K obtained from ClustalX

Phylogeny Tree

  • There are 2 main groups shown on the tree that has been constructed using results from BLASTP results, shown in the red is eukayotes and in the blue are all the bacterial species. Significant conservation among all the different species are seen suggesting sulfatases are members of an evolutionary conserved gene family sharing a common ancestor. However, the bacteria and lower eukaryotes show fewer sulfatase genes compared with higher eukaryotes such as homo sapiens. This may suggest a common ancestor was more closely related to sulfatases present today in lower sulfatases.

Figure 13
Evolutionary tree of proteins showing sequence homology with arylsulfatase K, red represents eukaryotes and blue represents bacteria