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

• DOI: 10.1073/pnas.1818333116
• Publication Date: 2019-03-15T00:35:32Z
• Authors: Claire Debarnot; Yoan R. Monneau; Véronique Roig-Za...; Vincent Delauzun; Christine Le Narvor; Emeline Richard; Jérôme Hénault; Adeline Goulet; Firas Fadel; Romain R. Vivès; Bernard Priem; David Bonnaffé; Hugues Lortat-Jacob; Yves Bourne
• Keywords: 0301 basic medicine; MESH: Heparin; [SDV.BIO]Life Sciences [q-bio]/Biotechnology; [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology; Molecular Biology/Biochemistry [q-bio.BM]; Oligosaccharides; catalytic mechanism; Crystallography, X-Ray; [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry; Catalysis; Substrate Specificity; Structure-Activity Relationship; 03 medical and health sciences; MESH: Structure-Activity Relationship; Glucuronic Acid; [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases; Humans; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM]; X-ray crystallography; [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases; MESH: Humans; Binding Sites; [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM]; Molecular Biology/Structural Biology [q-bio.BM]; Heparin; MESH: Crystallography; 500; MESH: Carbohydrate Epimerases; 540; MESH: Catalysis; MESH: Crystallography, X-Ray; [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM]; [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology; HEK293 Cells; [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry; MESH: Binding Sites; MESH: Glucuronic Acid; C5 epimerization; MESH: HEK293 Cells; [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology; X-Ray; [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology; MESH: Substrate Specificity; heparan sulfate; Carbohydrate Epimerases; MESH: Oligosaccharides; substrate distortion

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.