Interacting polymer-modification enzymes in heparan sulfate biosynthesis

Glucuronyl 5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) into L-iduronic acid (IdoA) units, through a mechanism involving reversible abstraction of a proton at C5 of hexuronic acid residues. Incubations of a [4GlcA beta 1-4GlcNSO3 alpha 1-]n precursor substrate with recombinant enzymes in a D2O/H2O medium enabled an isotope exchange approach to the assessment of functional interactions of Hsepi with hexuronyl 2-O-sulfotrans-ferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st), both involved in the final polymer-modification steps. Enzyme complexes were supported by computational modeling and homogeneous time resolved fluores-cence. GlcA and IdoA D/H ratios related to product composition revealed kinetic isotope effects that were interpreted in terms of efficiency of the coupled epimerase and sulfotransferase reactions. Evidence for a func-tional Hsepi/Hs6st complex was provided by selective incorporation of D atoms into GlcA units adjacent to 6-O -sulfated glucosamine residues. The inability to achieve simultaneous 2-O-and 6-O-sulfation in vitro supported topologically separated reactions in the cell. These findings provide novel insight into the roles of enzyme in-teractions in heparan sulfate biosynthesis.

Automated summary

This study investigated the functional interactions between glucuronyl 5-epimerase (Hsepi) and hexuronyl 2-O-sulfotransferase (Hs2st) and glucosaminyl 6-O-sulfotransferase (Hs6st) using an isotope exchange approach. The results showed that the binding of these enzymes plays an important role in the efficiency of substrate conversion. Furthermore, experimental evidence suggested the formation of a functional complex between Hsepi and Hs6st in cells, and the inability to achieve simultaneous 2-O and 6-O sulfation in vitro.