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The VanY_D DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK¹
Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France²

Author for correspondence: Peter E. Reynolds. Tel: +44 1223 333644. Fax: +44 1223 333345. e-mail: per{at}mole.bio.cam.ac.uk

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.

Keywords: glycopeptide resistance, VanD phenotype, membrane-bound DD-peptidase, vancomycin resistance

Abbreviations: PBP, penicillin-binding protein

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