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) 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)
Wu
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Wright
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Walsh
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004150:Dipeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D013379:Substrate Specificity; D014640:Vancomycin / Q000494:pharmacology*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009842:Oligopeptides; D014640:Vancomycin; D004150:Dipeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase