title: Bacterial resistance to vancomycin: overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a D-Ala-D-Ser ligase.
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).
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: I S
authors.initials: IS
authors.lastname: Park
authors.forename: C H
authors.initials: CH
authors.lastname: Lin
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
mesh_terms: D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D016983:Enterococcus / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014640:Vancomycin / Q000494:pharmacology*
chemical_list: D001426:Bacterial Proteins; D014640:Vancomycin; D010453:Peptide Synthases; C108395:VanC protein, Enterococcus