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Glycobiology. 2015 Mar;25(3):319-30. doi: 10.1093/glycob/cwu113. Epub 2014 Oct 24.

Structural and biochemical characterization of the β-N-acetylglucosaminidase from Thermotoga maritima: toward rationalization of mechanistic knowledge in the GH73 family.

Author information

1
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.
2
Laboratoire des Enveloppes Bactériennes et Antibiotiques, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR 8619 CNRS, Université de Paris-Sud, 91405 Orsay, France.
3
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.
4
European Synchrotron Radiation Facility, Polygone Scientifique Louis Néel, 6 rue Jules Horowitz, 38000 Grenoble, France.
5
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.

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.

KEYWORDS:

X-ray structure; catalytic mechanism; peptidoglycan; β-N-acetylglucosaminidase

PMID:
25344445
DOI:
10.1093/glycob/cwu113
[Indexed for MEDLINE]

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