Purification and characterization of VanXY C , a d , d ‐dipeptidase/ d , d ‐carboxypeptidase in vancomycin‐resistant Enterococcus gallinarum BM4174

VanXY C , a bifunctional enzyme from VanC‐phenotype Enterococcus gallinarum BM4174 that catalyses d , d ‐peptidase and d , d ‐carboxypeptidase activities, was purified as the native protein, as a maltose‐binding protein fusion and with an N‐terminal tag containing six histidine residues. The kinetic parameters of His 6 –VanXY C were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for d ‐Ala‐ d ‐Ala, the K m was 3.6 m m and k cat , 2.5 s −1 ; for UDP‐MurNAc‐L‐Ala‐ d ‐Glu‐L‐Lys‐ d ‐Ala‐ d ‐Ala (UDP‐MurNAc‐pentapeptide[Ala]), K m was 18.8 m m and k cat 6.2 s −1 ; for d ‐Ala‐ d ‐Ser, K m was 15.5 m m and k cat 0.35 s −1 . His 6 –VanXY C was inactive against the peptidoglycan precursor UDP‐MurNAc‐ l ‐Ala‐ d ‐Glu‐L‐Lys‐ d ‐Ala‐ d ‐Ser (UDP‐MurNAc‐pentapeptide[Ser]). The rate of hydrolysis of the terminal d ‐Ala of UDP‐MurNAc‐pentapeptide[Ala] was inhibited 30% by 2 m m d ‐Ala‐ d ‐Ser or UDP‐MurNAc‐pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in d ‐Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in d ‐Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan. Mutation of an aspartate residue (Asp59) of His‐tagged VanXY C corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant ( k cat / K m ) for d ‐Ala‐ d ‐Ala. This situation is in contrast to VanX in which mutation of Asp68→Ala produced a greater than 200 000‐fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.