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 vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid 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. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent d-Ala-d-Ser ligase 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. VanC2 also has substantial d-Ala-d-Asn ligase activity (k(cat)/K(m2) = 74 mM(−1)min(−1)).