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 and may be appropriate models for the mechanism of action of ASK. Only STS is fully functionally characterized. STS possesses 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 dimensional structure of ASK is not indicative of a transmembrane domain, so it is probably a water soluble enzyme found in ER [1].

Evidence from previous experiments state that ASA is localised in lysosomes [2]. The pH within lysosomes (5 - 5.5) is much lower than that found within the ER and golgi (neutral, find out) [3]. The water-soluble domain of STS is therefore likely to be a better model for the catalytic site of ASK. Nine key catalytic residues in STS are D35, D36, D342, G343, C75, R79, K134, K368, H136 and H290. The first four residues provide oxygen ligands for the divalent cation binding while others participate in hydrolysis of the substrate. It should be noted that cysteine is post translationally modified to a formylglycine (FG).

MSA revealed that these STS catalytic residues are conserved generally all through the enzyme family and especially in ASA. These residues were marked on the crystal structure of ASK using PyMol and they compose a very similar catalytic site to that of STS. Five out of nine STS key residues were found to be very similar to ASK, both in sequence alignment and in positioning within the active site. However D36 and C75 of STS seemed to have been replaced with histidine and serine respectively, while the role of STS-K368 appears to be replaced with K296 in ASK. 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 been conserved throughout the MSA.

As mentioned earlier, there are four residues which provide electronegative oxygen ligands to hold the divalent cation (for example, calcium or magnesium) 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. This is likely to be a conservative change as the histidine contains two nitrogen atoms whose lone pairs could form a coordinate bond with the divalent cation as can the oxygen atoms of aspartic acid.

Finally, the C75 of STS has been replaced by a serine in the bacterial ASK. This serine could easily be converted to a formylglycine. The difference between the serine and formylglycine funtional groups, a O.2A difference in bond length and the absence of two hydrogen atoms in formylglycine, cannot be differentiated at 2.4A crystal structure resolution. An experiment performed on Klebsiella pneumoneae’’,which expresses a very similar ASK to that of Bacteroides thetaiotaomicrone, revealed that the analogous serine residue is oxidised to formylglycine as a result of post translational modification of the polypeptide [[2]].

The C-terminus of sulfatases contains a substrate binding site and is 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 possible that ASK, like STS, hydrolyses steroid sulfates.