title: Structural and biochemical characterization of the β-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family.
abstract: Members of the GH73 glycosidase family cleave the β-1,4-glycosidic bond between the N-acetylglucosaminyl (GlcNAc) and N-acetylmuramyl (MurNAc) moieties in bacterial peptidoglycan. A catalytic mechanism has been proposed for members FlgJ, Auto, AcmA and Atl(WM) and the structural analysis of FlgJ and Auto revealed a conserved α/β fold reminiscent of the distantly related GH23 lysozyme. Comparison of the active site residues reveals variability in the nature of the catalytic general base suggesting two distinct catalytic mechanisms: an inverting mechanism involving two distant glutamate residues and a substrate-assisted mechanism involving anchimeric assistance by the C2-acetamido group of the GlcNAc moiety. Herein, we present the biochemical characterization and crystal structure of TM0633 from the hyperthermophilic bacterium Thermotoga maritima. TM0633 adopts the α/β fold of the family and displays β-N-acetylglucosaminidase activity on intact peptidoglycan sacculi. Site-directed mutagenesis identifies Glu34, Glu65 and Tyr118 as important residues for catalysis. A thorough bioinformatic analysis of the GH73 sequences identified five phylogenetic clusters. TM0633, FlgJ and Auto belong to a group of three clusters that conserve two carboxylate residues involved in a classical inverting acid-base mechanism. Members of the other two clusters lack a conserved catalytic general base supporting a substrate-assisted mechanism. Molecular modeling of representative members from each cluster suggests that variability in length of the β-hairpin region above the active site confers ligand-binding specificity and modulates the catalytic mechanisms within the GH73 family.
authors.affiliation: Laboratory for Biocrystallography and Structural Biology of Therapeutic Targets, Molecular and Structural Bases of Infectious Diseases, UMR 5086 CNRS and University of Lyon, 7 passage du Vercors, F-69367 Lyon Cedex 07, France CNRS, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lipski
authors.affiliation: Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université de Paris-Sud, 91405 Orsay, France.
authors.forename: Mireille
authors.initials: M
authors.lastname: Hervé
authors.affiliation: CNRS, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France Aix-Marseille University, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Lombard
authors.affiliation: European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, 6 rue Jules Horowitz, 38000 Grenoble, France.
authors.forename: Didier
authors.initials: D
authors.lastname: Nurizzo
authors.affiliation: Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université de Paris-Sud, 91405 Orsay, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mengin-Lecreulx
authors.affiliation: CNRS, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France Aix-Marseille University, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Bourne
authors.affiliation: CNRS, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France Aix-Marseille University, AFMB UMR7257, 163 avenue de luminy, 13288 Marseille cedex 09, France florence.vincent@afmb.univ-mrs.fr floyork13@gmail.com.
authors.forename: Florence
authors.initials: F
authors.lastname: Vincent
mesh_terms: D000118:Acetylglucosaminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D010802:Phylogeny; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D000118:Acetylglucosaminidase
keywords: X-ray structure; catalytic mechanism; peptidoglycan; β-N-acetylglucosaminidase