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), intol-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)nsubstrate or a (IdoA-GlcNS)nproduct. 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 ofN-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.

Heparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)‐N‐acetyl‐glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1–EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N‐deacetylase/N‐sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N‐deacetylation of the GlcNAc residues into GlcNH2 and its re‐N‐sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure‐driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N‐deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.