3bsqA Discussion

Multiple sequence alighment (MSA) revealed several conserved residues throughput the whole sulfatase family, perdominantly in the N-terminal region of the sequence. As mentioned in the introduction, ASA and ASB are lysosomal enzymes while ASC is a microsomal enzyme. Arylsulfatases D, E, F, G, H, J and K are localized in the ER and golgi compartments [3]. N-Acetylgalascosamine-4-sulfatase, ASA and STS were shown to be most similar in structure to ASK in 'DALI' results. Therefore the literature was searched to find the mechanism of catalytic action of these enzymes. Only STS is fully functionally characterised. STS posseses a set of nine residues which are essential for function. ([[1]] [1]). However STS is a membrane bound protein which consists of a globular domain bearing the catalytic site and a transmembrane domain made up of two antiparallel hydrophobic alpha helices. The three dimentional stucture of ASK is not indicative of a transmembrane domain; therefore it is probably a water soluble enzyme found in ER [1].

Evidence from previous experiments state that ASA is localysed in lysosomes [2], which has very low pH conditions to that of ER and golgi. Therefore STS was chosen to study the structure and function of the catalytic site. Nine key catalytic residues in STS are D35, D36, D342, G343, C75, R79, K134, K368, H136 and H290. First four residues provide oxygen ligands for the divalent cation binding while others participate in hydrolysis of the substrate. It should be noted that cystine is post translationally modified to a formyleglycine (FG).

MSA revealed that these STS catalytic residues are conserved generally all through the enzyme family and especially in ASA and STS. These residues were marked on the crystal structure of ASK on PyMol, they composes a very similar catalytic site to that of STS. Five out of nine STS key residues were found highly conserved in both sequence and crystal structure of ASK. However D36 and C75 of STS seemd to have replaced with histidine and serine , while STS-K368 equivalent aspartic acid occurred a few positions earlier and STS-H290 was shown lost from ASK in MSA. When these residues were marked in the crystal structure, a histidine was found in the same position of catalytic site as in STS, but occurred at position 284 of ASK sequence. It is marked in ‘cyan’ on figure 2 under 'results'. The STS-K368-like residue in ASK (K296) seemed to have conserved all throught the MSA. As mentionaed earlier, there are four residues which provide electronegative oxygen ligands to hold the divalent cation in the catalytic site. Three of these residues (D24, D283 and G284) are conserved in ASK, but D36 in STS is replaced by a histidine in ASK. However considering either of nitrogen on histidine side chain could donate a lone pair to form a coordinate bond with ‘the divalent cation’ this region may still function as the oxygrn ligand in aspartic acid. Finally, the C75 of STS has been replaced by a serine in the bacterial ASK, but this serine could easily be converted to a formylglycine. The crystal structure of ASK at 2.4 A resolution woudn't show the difference between a serine and a formylglycine, where only differences are lose of one hydrogen and formation of a double bond (1.2 A)instead of the single bond (1.4A) between carbon to oxygen. An experiment carried out on Klebsiella pneumoneae’’,which express a very similar ASK to that of Bacteroides thetaiotaomicrone, reveals that ASK polypeptides posseses FG in the place of serine as a result of a different post translational modification mechanism to that of eukaryotes [[2]].

The C-terminus of sulfatases contain a substrate binding site, hence weakly conserved throughout the family due to the variation of types of substrates used. However, when the C-terminal regions in two sequence alignment of ASK and STN was examined, the level of homology was very high (27.9%). Based on this evidence it is predicted, that STS and ASK shair substrates.

Possible function and likely substrates of ASK

STS is a membrane bound enzyme mostly found in the human placenta and skin fibroblasts. It converts sex-stroid precursors to ative estrogen and androgen, therefore provides a localised supply of these hormones (Ghosh, D., 2007). ASK is a water soluble enzyme localysed in ER of the cell. No tissue localization is specified to date. Based on all the above evidence, the possible function ASK may be binding and activation of stroids in the ER lumen.