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Free Content Gene vanXYC encodesd,d-dipeptidase (VanX) andd,d-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

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VanX and VanY have strictd,d-dipeptidase andd,d-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending ind-alanyl-d-alanine (d-Ala-d-Ala) in glycopeptide-resistant enterococci in which the C-terminald-Ala residue has been replaced byd-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending ind-Ala-d-serine (d-Ala-d-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis ofd-Ala-d-Ser has a polar effect on bothd,d-dipeptidase andd,d-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity againstd-Ala-d-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[d-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only whend-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis ofd-Ala-d-Ala and removal ofd-Ala from UDP-MurNAc-pentapeptide[d-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.
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Document Type: Research Article

Affiliations: 1: Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK., 2: Unité des Agents Antibactériens, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, Cedex 15, France.

Publication date: October 1, 1999

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