Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147

Biochemistry. 1995 Feb 28;34(8):2455-63. doi: 10.1021/bi00008a008.


Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Binding, Competitive
  • Cloning, Molecular
  • DNA Primers / genetics
  • DNA, Bacterial / genetics
  • Dipeptidases / genetics*
  • Dipeptidases / isolation & purification
  • Dipeptidases / metabolism
  • Drug Resistance, Microbial / genetics
  • Enterococcus faecium / drug effects
  • Enterococcus faecium / enzymology*
  • Enterococcus faecium / genetics*
  • Escherichia coli / genetics
  • Genes, Bacterial
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Oligopeptides / chemistry
  • Serine-Type D-Ala-D-Ala Carboxypeptidase*
  • Substrate Specificity
  • Vancomycin / pharmacology*


  • Bacterial Proteins
  • DNA Primers
  • DNA, Bacterial
  • Oligopeptides
  • Vancomycin
  • Dipeptidases
  • Serine-Type D-Ala-D-Ala Carboxypeptidase
  • VanX dipeptidase