Interacting polymer-modification enzymes in heparan sulfate biosynthesis

• Published in: Carbohydrate polymers 2023-01, Vol.299, p.120191-120191, Article 120191
• Main Authors: Zhang, Tianji, Yu, Mingjia, Li, Honglian, Maccarana, Marco, Zhang, Wei, Shi, Deling, Kan, Ying, Zhang, Xiao, Chi, Lianli, Lindahl, Ulf, Li, Hongmei, Li, Jin-ping, Tan, Tianwei
• Format: Article
• Language: English
• Subjects: 2-O-sulfotransferase; 6-Osulfotransferase; Glucuronic Acid; Glucuronosyl C5-epimerase; Heparan sulfate; Heparitin Sulfate; Iduronic Acid; Multienzyme Complexes; Polymers; Protons; Racemases and Epimerases; Sulfotransferases
• ISSN: 0144-8617; 1879-1344; 1879-1344
• DOI: 10.1016/j.carbpol.2022.120191

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 [4GlcAβ1–4GlcNSO3α1-]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-sulfotransferase (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 fluorescence. 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 functional 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 interactions in heparan sulfate biosynthesis. [Display omitted]