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 <i>N</i>-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.