Dissecting the substrate recognition of 3- O -sulfotransferase for the biosynthesis of anticoagulant heparin

Abstract

Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Sitedirected mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production.oligosaccharides | substrate specificity | ternary complex | heparan sulfate

Probing Structural Selectivity of Synthetic Heparin Binding to Stabilin Protein Receptors

“…We postulate that this effect is unlikely to be purely attributed to an increase in charge density because 3-Osulfation is a rare modification in HS (29). A recent study suggests that 3-O-sulfation may affect the conformation of neighboring IdoA2S residues to rearrange the positioning of sulfo groups (37). In addition, previous studies have shown that one natural highly sulfated chondroitin sulfate (CS-D) did not compete with heparin in contrast to other similar chondroitin sulfates (CS-B, CS-E) that demonstrated some degree of competition, providing evidence that charge is not the only determinant for heparin binding to the Stabilins (9).…”

Heparin Dodecasaccharide Containing Two Antithrombin-binding Pentasaccharides

“…3-O-sulfotransferase) in the Golgi apparatus, others suggest that the distribution of recognition sites for the 3-O-sulfotransferase may control the synthesis of the AT-binding site (28,31). Differences in structure and specificity of different 3-O-sulfotransferases, each of them exhibiting unique substrate recognition properties and distinct functional roles, may also influence the distribution of the AT-binding site in both physiological and pathophysiological conditions (9,32). At the present state of knowledge on the actual physiological function of heparin (26), the significance of biosynthesis of two contiguous active sites for AT remains elusive.…”

Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

“…The potential steric hindrance between the methyl group on residue 3 of this molecule (mimicking that of ristocetin) and Met102 at the N-terminal of the α5/α6 loop in StaL could be resolved by replacing Met102 with a Leu or Val. Notably, structure-guided mutagenesis of heparan sulfate 3-O-sulfotransferases at a similar location (the 3-OST-1 V164E mutant and the 3-OST-3 T256E mutant) resulted in mutants with higher substrate promiscuity and generated multiple sulfation products (22). Docking the modeled molecule within the StaL substrate-binding pocket also revealed additional unoccupied volume in the vicinity of the long fatty acyl moiety on residue 4 (SI Appendix, Fig.…”

“…Extensive structural analysis of eukaryotic sulfotransferases indicated that the substrate-binding site is formed mainly by three loops, corresponding to the loops/ regions α2/α3 (V1), α5/α6, and α12/α13 (V3) of StaL. Moreover, numerous studies also have shown that a change in substrate specificity for eukaryotic sulfotransferases could be achieved by mutations in these regions (20)(21)(22)(23). Thus, it is reasonable to assume that protein engineering by systematically modifying the residues in the foregoing regions (and thus the shape and property of the substrate-binding pocket) of these GPA sulfotransferases could lead to enzymes with different substrate regioselectivities.…”