Mechanism-Based Inactivation of VanX, a d-Alanyl-d-alanine Dipeptidase Necessary for Vancomycin Resistance

Abstract

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.

Substrate Inhibition of VanA byd-Alanine Reduces Vancomycin Resistance in a VanX-Dependent Manner

“…So far, attempts have included engineering VanX inhibitors (26,41,72) or reengineering vancomycin itself to target not only the cell wall precursors with D-Ala-D-Ala termini but also those ending with D-Ala-D-Lac (73). As a basis to develop new approaches to target vancomycin resistance, we studied the model organism S. coelicolor, which has a set of vancomycin resistance genes very similar to those of the pathogenic VRE (34).…”

“…The high sensitivity of vanX-null mutants to the combination of vancomycin and D-Ala strongly suggests that the combined treatment with vancomycin and D-Ala will be particularly effective in combination with molecules that perturb the bioactivity of VanX. VanX inhibitors have been described in the literature, but their effect was limited (26,41,72,78,79). Based on the data presented here, this is likely explained by the fact that the effect of a vanX deletion without additional D-Ala is very limited, decreasing the MIC by only 2-fold in this work.…”

“…Cells of E. coli were grown in Luria-Bertani broth (LB) at 37°C. Streptomyces coelicolor A3 (26) M145 was the parent of all mutants described in this work. All media and routine Streptomyces techniques were as described previously (47).…”

“…The VanA-type vancomycin resistance gene cluster in Streptomyces coelicolor consists of seven genes in four different operons, vanRS, vanJ, vanK, and vanHAX, which together mediate the substitution of the terminal D-alanine (D-Ala) by D-lactate (D-Lac), thereby decreasing the affinity of vancomycin for lipid II by three orders of magnitude (15,23). The vancomycin resistance gene cluster provides resistance to both vancomycin and teicoplanin and is located on the genome of the vancomycin producer Amycolatopsis mediterranei (24,25) as well as that of other actinomycetes, including the model species Streptomyces coelicolor A3 (26,27).…”

Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis

“…A thickened cell wall has been shown to be associated with vancomycin resistance in staphylococci (8). In the genus Enterococcus, resistance to vancomycin relies on the exchange of the terminal dipeptide in pentapeptide cell wall precursors (2). However, the molecular mechanisms of vancomycin resistance in mycobacteria are unknown.…”