Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase

Proc Natl Acad Sci U S A. 1997 Sep 16;94(19):10040-4. doi: 10.1073/pnas.94.19.10040.

Abstract

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 kcat/Km2 (Km2 = Km 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 (kcat/Km2 = 74 mM-1min-1).

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins*
  • Cloning, Molecular
  • Drug Resistance, Microbial / genetics
  • Enterococcus / enzymology*
  • Escherichia coli / genetics
  • Peptide Synthases / genetics*
  • Peptide Synthases / isolation & purification
  • Peptide Synthases / metabolism
  • Vancomycin / pharmacology*

Substances

  • Bacterial Proteins
  • Vancomycin
  • Peptide Synthases
  • VanC protein, Enterococcus