Purification and characterization of VanXYC, 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 mm 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 mm and k (cat) 6.2 s(-1) ; for d-Ala-d-Ser, K (m) was 15.5 mm 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 mm 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.