OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATION OF VANX, A D-,D-DIPEPTIDASE WHICH IS ESSENTIAL FOR VANCOMYCIN RESISTANCE IN ENTEROCOCCUS-FAECIUM-BM4147

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) Mel. 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 k(cat) and K-m 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, k(cat) 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 (K-i estimated at 242 mM) but also due to a k(cat) 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. Phosphinate analogues of the proposed tetrahedral adduct for hydrolysis of D-Ala-D-Ala show mixed-type noncompetitive inhibition of VanX at K-is of approximately 0.4 mu M, for a K-m/K-is ratio of 2500:1. The dipeptidase activity of VanX may be a target for drug design to reverse clinical vancomycin resistance.