Requirement of the VanY and VanX D,D‐peptidases for glycopeptide resistance in enterococci

Transposon Tn1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D‐peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N‐terminal membrane anchor of the protein. The enzyme was a Zn2+‐dependent D,D‐carboxypeptidase that cleaved the C‐terminal residue of peptidoglycan precursors ending in R‐D‐Ala‐D‐Ala or R‐D‐Ala‐D‐Lac but not the dipeptide D‐Ala‐D‐Ala. The specificity constants kcat/Km were 17‐ to 67‐fold higher for substrates ending in the R‐D‐Ala‐D‐Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP‐MurNAc‐tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high‐level glycopeptide resistance in a medium supplemented with D‐Ala. The enzyme could not replace the VanX D,D‐dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D‐Ala:D‐Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D‐carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D‐Ala‐containing medium. Thus, VanX and VanY had non‐overlapping functions involving the hydrolysis of D‐Ala‐D‐Ala and the removal of D‐Ala from membrane‐bound lipid intermediates respectively.