title: The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein.
abstract: VanD-type Enterococcus faecium BM4339 is constitutively resistant to vancomycin and to low levels of teicoplanin. This strain produces peptidoglycan precursors terminating in D-lactate but, unlike VanA- and VanB-type strains, E. faecium BM4339 has a mutated ddl ligase gene and cannot synthesize D-Ala-D-Ala. Consequently, although it possesses vanX(D) and vanY(D) genes, it should not require an active VanX-type DD-dipeptidase or a VanY-type DD-carboxypeptidase for resistance. The vanY(D) gene contains the signatures of a penicillin-binding protein (PBP) and is believed to encode a penicillin-sensitive DD-carboxypeptidase. The enzyme activity was found to be membrane-bound and inhibited by low concentrations of benzylpenicillin in membrane preparations and in intact bacteria, indicating that the active site was present on the outside surface of the membrane. The 38 kDa protein was revealed as a PBP present in more copies per cell than conventional PBPs and all the protein was accessible to benzylpenicillin added externally, confirming the localization of the active site. A glycopeptide-susceptible strain of E. faecium lacked this PBP, and the membrane-bound DD-carboxypeptidase activity was less than 5% of that of E. faecium BM4339. Although the active site of VanY(D) was external to the membrane, UDP-MurNAc-tetrapeptide was produced internally, probably from UDP-MurNAc-pentadepsipeptide. The presence of benzylpenicillin at low concentrations in the growth medium substantially reduced the amount of tetrapeptide produced, indicating that inhibition of VanY(D) by benzylpenicillin influenced production of peptidoglycan precursors internally. A model to explain these contrasting observations is proposed.
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK1.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Reynolds
authors.affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK1.
authors.forename: O Herman
authors.initials: OH
authors.lastname: Ambur
authors.affiliation: Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France2.
authors.forename: Barbara
authors.initials: B
authors.lastname: Casadewall
authors.affiliation: Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France2.
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D016984:Enterococcus faecium / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006602:Hexosyltransferases; D008565:Membrane Proteins; D008954:Models, Biological; D002267:Muramoylpentapeptide Carboxypeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D010400:Penicillin G / Q000494:pharmacology; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000378:metabolism; D010458:Peptidyl Transferases; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D020713:Vancomycin Resistance
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009842:Oligopeptides; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D014538:Uridine Diphosphate N-Acetylmuramic Acid; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002268:Carboxypeptidases; C117912:VanY protein, Bacteria; D002267:Muramoylpentapeptide Carboxypeptidase; D010400:Penicillin G