D-Ala–d-X ligases: evaluation of d-alanyl phosphate intermediate by MIX, PIX and rapid quench studies

Abstract

Background: The d-alanyl–d-lactate (d-Ala–d-Lac) ligase is required for synthesis of altered peptidoglycan (PG) termini in the VanA phenotype of vancomycin-resistant enterococci (VRE), and the d-alanyl–d-serine (d-Ala–d-Ser) ligase is required for the VanC phenotype of VRE. Here we have compared these with the Escherichia colid-Ala–d-Ala ligase DdlB for formation of the enzyme-bound d-alanyl phosphate, d-Ala1-PO32– (d-Ala1-P), intermediate.Results: The VanC2 ligase catalyzes a molecular isotope exchange (MIX) partial reaction, incorporating radioactivity from 14C-d-Ser into d-Ala–14C-d-Ser at a rate of 0.7 min−1, which approaches kinetic competence for the reversible d-Ala1-P formation from the back direction. A positional isotope exchange (PIX) study with the VanC2 and VanA ligases displayed a d-Ala1-dependent bridge to nonbridge exchange of the oxygen-18 label of [γ-18O4]-ATP at rates of up to 0.6 min−1; this exchange was completely suppressed by the addition of the second substrate d-Ser or d-Lac, respectively, as the d-Ala1-P intermediate was swept in the forward direction. As a third criterion for formation of bound d-Ala1-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB and VanA. With E. coli DdlB, there was a burst amplitude of ADP corresponding to 26–30% of the DdlB active sites, followed by the expected steady-state rate of 620–650 min−1. For d-Ala–d-Lac and d-Ala–d-Ala synthesis by VanA, we measured a burst of 25–30% or 51% of active enzyme, respectively.Conclusions: These three approaches support the rapid (more than 1000 min−1), reversible formation of the enzyme intermediate d-Ala1-P by members of the d-Ala–d-X (where X is Ala, Ser or Lac) ligase superfamily.