New chromogenic dipeptide substrate for continuous assay of the d‐alanyl‐d‐alanine dipeptidase VanX required for high‐level vancomycin resistance

Abstract

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.

Identification of (S,S)-γ-Glutamyl-(cis-S-1-propenyl)thioglycine, a Naturally Occurring Norcysteine Derivative, from the Chinese Vegetable Toona sinensis

“…This intermediate rapidly hydrolyzes to release ammonia, a thiol, and glyoxylic acid (Figure ) . Synthetic compounds of this type have been studied as delivery systems for antimicrobial compounds and as probes for peptidase activity. − …”

Specificity of Induction of the vanA and vanB Operons in Vancomycin-Resistant Enterococci by Telavancin

“…An endpoint assay of VanX D,D-dipeptidase activity coupled to D-amino acid oxidase and peroxidase has been utilized as a sensitive measure of the induction of the vancomycin resistance operon in Enterococcus faecalis (2,5). More recently, a continuous assay using the alternative peptide substrate D-alanyl-(R)-phenylthioglycine has been developed to assay VanX activity (1).…”