title: Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm.
abstract: In Mycobacterium tuberculosis and ampicillin-resistant mutants of Enterococcus faecium, the classical target of β-lactam antibiotics is bypassed by L,D-transpeptidases that form unusual 3 → 3 peptidoglycan cross-links. β-lactams of the carbapenem class, such as ertapenem, are mimics of the acyl donor substrate and inactivate l,d-transpeptidases by acylation of their catalytic cysteine. We have blocked the acyl donor site of E. faecium L,D-transpeptidase Ldt(fm) by ertapenem and identified the acyl acceptor site based on analyses of chemical shift perturbations induced by binding of peptidoglycan fragments to the resulting acylenzyme. An nuclear magnetic resonance (NMR)-driven docking structure of the complex revealed key hydrogen interactions between the acyl acceptor and Ldt(fm) that were evaluated by site-directed mutagenesis and development of a cross-linking assay. Three residues are reported as critical for stabilisation of the acceptor in the Ldt(fm) active site and proper orientation of the nucleophilic nitrogen for the attack of the acylenzyme carbonyl. Identification of the catalytic pocket dedicated to the acceptor substrate opens new perspectives for the design of inhibitors with an original mode of action that could act alone or in synergy with β-lactams.
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Equipe 12, F-75006, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Triboulet
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|CNRS, IBS, F-38044 Grenoble, France.|CEA, IBS, F-38044 Grenoble, France.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Bougault
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|CNRS, IBS, F-38044 Grenoble, France.|CEA, IBS, F-38044 Grenoble, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Laguri
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Equipe 12, F-75006, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.
authors.forename: Jean-Emmanuel
authors.initials: JE
authors.lastname: Hugonnet
authors.affiliation: INSERM, UMR_S 1138, Centre de Recherche des Cordeliers, Equipe 12, F-75006, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, F-75006, Paris, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Arthur
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|CNRS, IBS, F-38044 Grenoble, France.|CEA, IBS, F-38044 Grenoble, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Simorre
mesh_terms: D000215:Acylation; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D016984:Enterococcus faecium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D000077727:Ertapenem; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D010458:Peptidyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D010457:Peptidoglycan; D047090:beta-Lactams; D010458:Peptidyl Transferases; D000077727:Ertapenem