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The VanY(D) DD-carboxypeptidase of Enterococcus faecium BM4339 is a penicillin-binding protein
Peter E Reynolds1, O Herman Ambur1, Barbara Casadewall2
1Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK1.
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.
Insights
VanD-type Enterococcus faecium BM4339 exhibits vancomycin resistance due to a mutated ddl ligase gene. Its VanY(D) protein, a penicillin-binding protein, is membrane-bound and externally located, influencing peptidoglycan precursor synthesis.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- VanD-type Enterococcus faecium BM4339 displays intrinsic resistance to vancomycin and teicoplanin.
- This strain synthesizes peptidoglycan precursors ending in D-lactate and possesses a mutated ddl ligase gene, preventing D-Ala-D-Ala synthesis.
Purpose of the Study:
- To investigate the role of the vanY(D) gene and its encoded protein in the vancomycin resistance mechanism of E. faecium BM4339.
- To characterize the enzymatic activity and cellular localization of the VanY(D) protein.
Main Methods:
- Enzyme activity assays using membrane preparations and intact bacteria.
- Inhibition studies with benzylpenicillin.
- Protein characterization including molecular weight determination and accessibility studies.
- Comparison with a glycopeptide-susceptible E. faecium strain.
Main Results:
- The vanY(D) gene encodes a membrane-bound DD-carboxypeptidase with an external active site, identified as a penicillin-binding protein (PBP).
- This PBP is present in higher quantities than conventional PBPs and is sensitive to low concentrations of benzylpenicillin.
- E. faecium BM4339 exhibits significantly higher DD-carboxypeptidase activity compared to susceptible strains.
- Benzylpenicillin inhibition of VanY(D) impacts internal peptidoglycan precursor production.
Conclusions:
- The VanY(D) protein, a PBP with an external active site, plays a crucial role in the vancomycin resistance of E. faecium BM4339.
- Its interaction with benzylpenicillin influences peptidoglycan precursor synthesis, offering insights into resistance mechanisms.
- A model is proposed to explain the observed resistance and precursor synthesis pathway.