title: Structural and functional characterization of the Clostridium perfringens N-acetylmannosamine-6-phosphate 2-epimerase essential for the sialic acid salvage pathway.
abstract: Pathogenic bacteria are endowed with an arsenal of specialized enzymes to convert nutrient compounds from their cell hosts. The essential N-acetylmannosamine-6-phosphate 2-epimerase (NanE) belongs to a convergent glycolytic pathway for utilization of the three amino sugars, GlcNAc, ManNAc, and sialic acid. The crystal structure of ligand-free NanE from Clostridium perfringens reveals a modified triose-phosphate isomerase (β/α)8 barrel in which a stable dimer is formed by exchanging the C-terminal helix. By retaining catalytic activity in the crystalline state, the structure of the enzyme bound to the GlcNAc-6P product identifies the topology of the active site pocket and points to invariant residues Lys(66) as a putative single catalyst, supported by the structure of the catalytically inactive K66A mutant in complex with substrate ManNAc-6P. (1)H NMR-based time course assays of native NanE and mutated variants demonstrate the essential role of Lys(66) for the epimerization reaction with participation of neighboring Arg(43), Asp(126), and Glu(180) residues. These findings unveil a one-base catalytic mechanism of C2 deprotonation/reprotonation via an enolate intermediate and provide the structural basis for the development of new antimicrobial agents against this family of bacterial 2-epimerases.
authors.affiliation: From the Aix-Marseille University, AFMB UMR7257, 163 avenue de Luminy 13288 Marseille, France, the CNRS, AFMB UMR7257, 163 avenue de Luminy, 13288 Marseille, France.
authors.forename: Marie-Cécile
authors.initials: MC
authors.lastname: Pélissier
authors.affiliation: the Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS UMR7255, Aix-Marseille Université, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, and.
authors.forename: Corinne
authors.initials: C
authors.lastname: Sebban-Kreuzer
authors.affiliation: the Laboratoire d'Ingénierie des Systèmes Macromoléculaires, CNRS UMR7255, Aix-Marseille Université, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France, and.
authors.forename: Françoise
authors.initials: F
authors.lastname: Guerlesquin
authors.affiliation: the Department of Chemistry, Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Brannigan
authors.affiliation: From the Aix-Marseille University, AFMB UMR7257, 163 avenue de Luminy 13288 Marseille, France, the CNRS, AFMB UMR7257, 163 avenue de Luminy, 13288 Marseille, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Bourne
authors.affiliation: From the Aix-Marseille University, AFMB UMR7257, 163 avenue de Luminy 13288 Marseille, France, the CNRS, AFMB UMR7257, 163 avenue de Luminy, 13288 Marseille, France, florence.vincent@afmb.univ-mrs.fr.
authors.forename: Florence
authors.initials: F
authors.lastname: Vincent
mesh_terms: D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D018360:Crystallography, X-Ray; D006595:Hexosamines / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006595:Hexosamines; C569296:N-acetylmannosamine 6-phosphate; D011522:Protons; D013403:Sugar Phosphates; C026146:N-acetylglucosamine-1-phosphate; D002238:Carbohydrate Epimerases; C120657:N-acetylmannosamine-6-phosphate epimerase; D019158:N-Acetylneuraminic Acid; D008239:Lysine; D000117:Acetylglucosamine
keywords: 1H NMR Spectroscopy; Carbohydrate; Enzyme Mechanism; Mutagenesis; Sialic Acid; Sugar 2-Epimerase; Sugar Phosphate; X-ray Crystallography