Homologs of the vancomycin resistance d-Ala-d-Ala dipeptidase VanX in Streptomyces toyocaensis, Escherichia coli and Synechocystis: attributes of catalytic efficiency, stereoselectivity and regulation with implications for function

AbstractBackground: Vancomycin-resistant enterococci are pathogenic bacteria that have altered cell-wall peptidoglycan termini (d-alanyl-d-lactate [d-Ala-d-lactate] instead of d-alanyl-d-alanine [d-Ala-d-Ala]), which results in a 1000-fold decreased affinity for binding vancomycin. The metallodipeptidase VanX (EntVanX) is a key enzyme in antibiotic resistance as it reduces the cellular pool of the d-Ala-d-Ala dipeptide.Results: A bacterial genome search revealed vanX homologs in Streptomyces toyocaensis (StoVanX), Escherichia coli (EcoVanX), and Synechocystis sp. strain PCC6803 (SynVanX). Here, the d,d-dipeptidase catalytic activity of all three VanX homologs is validated, and the catalytic efficiencies and diastereoselectivity ratios for dipeptide cleavage are reported. The ecovanX gene is shown to have an RpoS (σs)-dependent promoter typical of genes turned on in stationary phase. Expression of ecovanX and an associated cluster of dipeptide permease genes permitted growth of E. coli using d-Ala-d-Ala as the sole carbon source.Conclusions: The key residues of the EntVanX active site are strongly conserved in the VanX homologs, suggesting their active-site topologies are similar. StoVanX is a highly efficient d-Ala-d-Ala dipeptidase; its gene is located in a vanHAX operon, consistent with a vancomycin-immunity function. StoVanX is a potential source for the VanX found in gram-positive enterococci. The catalytic efficiencies of d-Ala-d-Ala hydrolysis for EcoVanX and SynVanX are 25-fold lower than for EntVanX, suggesting they have a role in cell-wall turnover. Clustered with the ecovanX gene is a putative dipeptide permease system that imports d-Ala-d-Ala into the cell. The combined action of EcoVanX and the permease could permit the use of d-Ala-d-Ala as a bacterial energy source under starvation conditions