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Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase

Abstract : Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.
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Submitted on : Monday, December 21, 2020 - 4:04:19 PM
Last modification on : Tuesday, June 14, 2022 - 10:41:46 AM


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Claire Debarnot, Yoan Monneau, Veronique Roig-Zamboni, Vincent Delauzun, Christine Le Narvor, et al.. Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase. Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2019, 116 (14), pp.6760-6765. ⟨10.1073/pnas.1818333116⟩. ⟨hal-02339215⟩



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