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

Background: The D-alanyl-D-lactate (D-Ala-D-Lac) ligase is required for syn thesis of altered peptidoglycan (PG) termini in the VanA phenotype of vanco mycin-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 the se with the Escherichia coli D-Ala-D-Ala ligase DdlB for formation of the e nzyme-bound D-alanyl phosphate, D-Ala(1)-PO32- (D-Ala(1)-P), intermediate. Results: The VanC2 ligase catalyzes a molecular isotope exchange (MIX) part ial reaction, incorporating radioactivity from C-14-D-Ser into D-Ala-C-14-D -Ser at a rate of 0.7 min(-1),which approaches kinetic competence for the r eversible D-Ala(1)-P formation from the back direction. A positional isotop e exchange (PIX) study with the VanC2 and VanA ligases displayed a D-Ala(1) -dependent bridge to nonbridge exchange of the oxygen-18 label of [gamma-O- 18(4)]-ATP at rates of up to 0.6 min(-1); this exchange was completely supp ressed by the addition of the second substrate D-Ser or D-Lac, respectively , as the D-Ala(1)-P intermediate was swept in the forward direction. As a t hird criterion for formation of bound D-Ala(1)-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB an d 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-Ala(1)-P by members of the D-Ala-D-X (where X is Ala, Ser or Lac) ligase superfamily.