3bsqA Discussion

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Structural analysis of arylsulfatase K

Multiple sequence alighment (MSA) revealed several conserved residues throughout the whole sulfatase family, predominantly 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 [4]. 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 [4]. 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 [4,5].

Evidence from previous experiments state that ASA binds membrane lipid sulfatids and is localised in lysosomes [2]. The pH within lysosomes (5 - 5.5) is much lower than that found within the ER and golgi, which is neutrl. [4]. The water-soluble domain of STS is therefore likely to be a better model for the catalytic site of ASK. Nine out of ten key catalytic residues in STS are conserved with ASA and ASB [4]. These residues of STS are D35, D36, D342, G343, C75, R79, K134, K368, H136 and H290 (Figure 6) [5]. 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) [2,4].

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 purple on 'figure: 7' 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 0.2Å difference in bond length and the absence of two hydrogen atoms in formylglycine, cannot be differentiated at 2.4Å 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. A certain group of bacteria including two of above mentioned strains use a different post translational modification system in this process. See 'evolution of sulfatases' for details.

Possible function and likely substrates of ASK

STS is a membrane bound enzyme, mostly found in human placenta and skin fibroblasts. It converts sex-stroid precursors to ative estrogen and androgen, thereofre give a local suppley of these hormones [4]. Its catalytic site is buried deep with in the molecule and only connected to the exterior via a substrate-entry path. ASA is a water soluble enzyme found in lysosomes and 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. ASK structure also show number of hydrophobic residues on the surface (Figure 11), possibly indicating the hydrophobic nature of the substrate it binds.

Evolution of sulfatases

The phylogeny tree shows evidence that sulfatases are found in species of Bacteria and Eukaryotes. Few of the lower Eukaryotes, Archaea and most plant species lack sulfatases. The significant sequence conservation among different species suggests that sulfatases are members of an evolutionary conserved gene family sharing a common ancestor. Bacteria and lower eukaryotes have fewer sulfatase genes compared with the higher eukaryotes such as humans, suggesting that a common ancestor was more closely related to sulfatases present today in lower sulfatases.

Previous phylogeny studies have found decades ago the sulfatase family underwent a posttranslational modification which is vital for their enzymatic activity. Modifications involved highly conserved cysteine located in the active site of sulfatase, which is modified into formylglycine (FG). The modification factor of cysteine is SUMF1. This gene is highly conserved across species including prokaryotes to eukaryotes ranging from bacteria, fruit flies to mammals. It has been determined that species containing SUMF1 will also contain sulfatases in their genome, this suggests that the sulfatases are targets for this posttranslational modification. SUMF1 and SUMF2 have been identified, however only in vertebrates. Analysis of the evolution of these two factors suggests that SUMF2 evolved independently of SUMF1.

In addition, it has been found that some bacteria have been identified with serine in place of cysteine and this has been true for ASK. As well as cysteine, serine also undergoes modification into FG, however another modifying factor named AtsB is used rather than SUMF1, which are found not be related. Studies show most bacteria containing SUMF1 genes will side with cysteine-type sulfatase, whereas the AtsB gene will side with both cysteine and serine-type sulfatases, suggesting that AstB modifies both types of sulfatases. Evolutionary analysis proposes the first type of sulfatase was in fact cysteine, which also coevolved with SUMF1 modifier. Later serine evolved with the AstB modifier, which in turn transferred to other bacteria through horizontal transfer.

Multiple sequence alignment has shown species containing ASB, ASA, ASC and glucosamine-6-sulfatase show similarity extending over entire sequences, especially observed in the N- terminal, which composes one third of the protein. More specifically, conserved amino acid regions containing arginine and histidine residues. Peters (1990) determined histidine and arginine are essential for the catalytic activity in ASA suggesting that the conserved amino acid regions are involved in the assembly of the active sites of all four arylsulfatases.