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Glycopeptide resistance mediated by enterococcal transposon Tn 1546 requires production of VanX for hydrolysis of D‐alanyl‐D‐alanine

Cloning and nucleotide sequencing indicated that transposon Tn 1546 from Enterococcus faecium BM4147 encodes a 23365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2‐2 derivative producing VanH, VanA and VanX accumulated mainly UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Lac (pentadepsipeptide) and small amounts of UDP‐MurNAc‐L‐Ala‐γ‐D‐Glu‐L‐Lys‐D‐Ala‐D‐Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D‐dipeptidase that hydrolysed D‐Ala‐D‐Ala. Pentadepsipeptide, pentapeptide and D‐Ala‐D‐Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D‐dipeptidase that hydrolyses D‐Ala‐D‐Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D‐Ala‐D‐Ala‐containing peptidoglycan precursors at the cell surface.