Open AccessBacterial resistance to vancomycin: Overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a d -Ala- d -Ser ligase
Author(s) -
IlSeon Park,
ChunHung Lin,
Christopher T. Walsh
Publication year - 1997
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.94.19.10040
Subject(s) - overproduction , enterococcus , microbiology and biotechnology , vancomycin , chemistry , dna ligase , bacteria , biochemistry , biology , antibiotics , enzyme , genetics , staphylococcus aureus
The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited byEnterococcus gallinarum andEnterococcus casseliflavus . Based on the detection of the cell precursor UDP-N -acetylmuramic acid pentapeptide intermediate terminating ind -Ala-d -Ser instead ofd -Ala-d -Ala, it has been predicted that the VanC ligase would be ad -Ala-d -Ser rather than ad -Ala-d -Ala ligase. Overproduction of theE. casseliflavus ATCC 25788vanC2 gene inEscherichia coli and its purification to homogeneity allowed demonstration of ATP-dependentd -Ala-d -Ser ligase activity. Thek cat /K m2 (K m2 =K m ford -Ser or C-terminald -Ala) ratio ford -Ala-d -Ser/d -Ala-d -Ala dipeptide formation is 270/0.69 for a 400-fold selection againstd -Ala in the C-terminal position. VanC2 also has substantiald -Ala-d -Asn ligase activity (k cat /K m2 = 74 mM−1 min−1 ).