Gene vanXY(c) encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity , respectively, that eliminates production of peptidoglycan precursors endi ng in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococc i in which the C-terminal D-Ala residue has been replaced by D-lactate. Ent erococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catal yses synthesis of D-Ala-D-Ser has a polar effect on both D,D-dipeptidase an d D,D-carboxypeptidase activities. The open reading frame downstream from v anC-1 encoded a soluble protein designated VanXY(C) (M-r 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY i n an overlap of 158 amino acids, and contained consensus sequences for bind ing zinc, stabilizing the binding of substrate and catalysing hydrolysis th at are present in both VanX- and VanY-type enzymes, It had very low dipepti dase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP -MurNAc-pentapeptide[D-Ser], unlike VanY, The introduction of plasmid pAT70 8(vanC-1,XYC) or pAT717(vanXY(C)) into vancomycin-susceptible Enterococcos faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser wa s 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 funct ion of VanXY(C) was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP -MurNAc-pentapeptide[D-Ala], VanC-1 and VanXY(C) were essential, but not su fficient, for vancomycin resistance.