BACTERIAL-RESISTANCE TO VANCOMYCIN - OVERPRODUCTION, PURIFICATION, AND CHARACTERIZATION OF VANC2 FROM ENTEROCOCCUS-CASSELIFLAVUS AS A D-ALA-D-SER LIGASE

The VanC phenotype for clinical resistance of enterococci to vancomyci n is exhibited by Enterococcus gallinarum and Enterococcus casseliflav us. Based on the detection of the fell precursor UDP-N-acetylmuramic a cid pentapeptide intermediate terminating in D-Ala-D-Ser instead of D- Ala-D-Ala, it has been predicted that the VanC ligase would be a D-Ala -D-Ser rather than a D-Ala-D-Ala ligase, Overproduction of the E. cass eliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purificati on to homogeneity allowed demonstration of ATP dependent D-Ala-D-Ser l igase activity, The k(cat)/K-m2 (K-m2 = K-m for D-Ser or C-terminal D- Ala) ratio for D-Ala-D-Ser/D-Ala-D-Ala dipeptide formation is 270/0.69 for a 400-fold selection against D-Ala in the C-terminal position. Va nC2 also has substantial D-Ala-D-Asn ligase activity (k(cat)/K-m2 = 74 mM-(1)min(-1)).