Real-time monitoring of D-Ala-D-Ala dipeptidase activity of VanX in living bacteria by isothermal titration calorimetry.

The d,d-dipeptidase enzyme VanX is the main cause of vancomycin resistance in gram-positive bacteria because of hydrolysis of the D-Ala-D-Ala dipeptide used in cell-wall biosynthesis. Continuous assay of VanX has proven challenging due to lack of a chromophoric substrate. Here, we report a direct approach for continuous assay of VanX in vitro and in vivo from hydrolysis of D-Ala-D-Ala, based on the heat-rate changes measured with isothermal titration calorimetry (ITC). With the ITC approach, determination of kinetic parameters of VanX hydrolyzing D-Ala-D-Ala and the inhibition constant of d-cysteine inhibitor yielded KM of 0.10 mM, kcat of 11.5 s-1, and Ki of 18.8 µM, which are consistent with the data from ninhydrin/Cd(II) assays. Cell-based ITC studies demonstrated that the VanX expressed in E. coli and in clinical strain VRE was inhibited by d-cysteine with IC50 values of 29.8 and 28.6 µM, respectively. Also, the total heat from D-Ala-D-Ala (4 mM) hydrolysis decreases strongly (in absolute value) from 1.26 mJ for VRE to 0.031 mJ for E. faecalis, which is consistent with the large MIC value of vancomycin of 512 µg/mL for VRE and the much smaller value of 4 µg/mL for E. faecalis. The ITC approach proposed here could be applied to screen and evaluate small molecule inhibitors of VanX or to identify drug resistant bacteria.

Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.

As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.