Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

Biochemistry. 1991 Oct 29;30(43):10408-15. doi: 10.1021/bi00107a007.


Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Carbon-Oxygen Ligases*
  • Drug Resistance, Microbial / genetics
  • Enterococcus faecium / drug effects*
  • Enterococcus faecium / genetics
  • Genes, Bacterial
  • Kinetics
  • L-Lactate Dehydrogenase / metabolism
  • Molecular Sequence Data
  • Protein Precursors / biosynthesis
  • Sequence Alignment
  • Substrate Specificity
  • Vancomycin / metabolism
  • Vancomycin / pharmacology*


  • Bacterial Proteins
  • Protein Precursors
  • VanA ligase, Bacteria
  • VanH protein, Enterococcus faecium
  • Vancomycin
  • L-Lactate Dehydrogenase
  • Carbon-Oxygen Ligases