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

descriptionPublicationkeyboard_double_arrow_right Article 14 Mar 2019 English Publisher:Proceedings of the National Academy of SciencesJournal:Proceedings of the National Academy of Sciences, volume 116, pages 6,760-6,765 (issn: 0027-8424, eissn: 1091-6490, Copyright policy )
Authors: Debarnot, Claire; Monneau, Yoan; Roig-Zamboni, Veronique; Delauzun, Vincent; Le Narvor, Christine; Richard, Emeline; Hénault, Jérôme; +7 Authors

doi: 10.1073/pnas.1818333116

pmid: 30872481

pmc: PMC6452739

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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Keywords

MESH: Heparin, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Oligosaccharides, catalytic mechanism, Crystallography, X-Ray, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Substrate Specificity, MESH: Structure-Activity Relationship, Glucuronic Acid, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, [SDV.BBM.BC] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.MHEP.ME] Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.MP.VIR] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Crystallography, MESH: Carbohydrate Epimerases, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: Glucuronic Acid, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, MESH: HEK293 Cells, [SDV.SP.PHARMA] Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, heparan sulfate, substrate distortion, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Molecular Biology/Biochemistry [q-bio.BM], Catalysis, Structure-Activity Relationship, Humans, [SDV.IMM.II] Life Sciences [q-bio]/Immunology/Innate immunity, X-ray crystallography, MESH: Humans, Binding Sites, Molecular Biology/Structural Biology [q-bio.BM], Heparin, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, 500, MESH: Catalysis, 540, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, HEK293 Cells, MESH: Binding Sites, C5 epimerization, X-Ray, [SDV.SP.PHARMA]Life Sciences [q-bio]/Pharmaceutical sciences/Pharmacology, MESH: Substrate Specificity, [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Carbohydrate Epimerases, MESH: Oligosaccharides

  • BIP!
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    This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    		 33
    popularity
    This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
    		 Top 10%
    influence
    This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
    		 Average
    impulse
    This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
    		 Top 10%
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citations
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
33
Top 10%
Average
Top 10%
Green
bronze
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