Substrate binding mode and catalytic mechanism of human heparan sulfate d-glucuronyl C5 epimerase - Groupe Structure et Activité des Glycosaminoglycanes / Structure and Activity of Glycosaminoglycans Group (IBS-SAGAG) Accéder directement au contenu
Article Dans Une Revue Proceedings of the National Academy of Sciences of the United States of America Année : 2019

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

Romain R Vivès
Hugues Lortat-Jacob

Résumé

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.
Fichier principal
Vignette du fichier
6760.full.pdf (1.25 Mo) Télécharger le fichier
Origine : Fichiers produits par l'(les) auteur(s)

Dates et versions

hal-02339215 , version 1 (21-12-2020)

Licence

Paternité - Pas d'utilisation commerciale - Pas de modification

Identifiants

Citer

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, 2019, 116 (14), pp.6760-6765. ⟨10.1073/pnas.1818333116⟩. ⟨hal-02339215⟩
240 Consultations
74 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More