Determinants for Differential Effects on d-Ala-d-Lactate vs d-Ala-d-Ala Formation by the VanA Ligase from Vancomycin-Resistant Enterococci

Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-d-Ala-d-Ala termini that are the antibiotic-binding target to either PG-d-Ala-d-lactate or PG-d-Ala-d-Ser as a consequence of altered specificity of the d-Ala-d-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a d-Ala-d-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile d-lactate selectively over d-Ala2 to capture the d-Ala1-OPO3 2- intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH3 +) form of d-Ala at subsite 2 (K M2 of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH2) form of d-Ala (K M2 of 0.66 mM, k cat of 550 min-1) is a 17-fold better substrate compared to d-lactate (K M of 0.69 mM, k cat of 32 min-1). The low concentration of the free amine form of d-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative ω-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of d-Ala2. The VanA enzyme represents a subfamily of d-Ala-d-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site ω-loop of the Escherichia coli d-Ala-d-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both d-Ala-d-Ala dipeptide and d-Ala-d-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K M2 for d-lactate but not d-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile d-lactate. Replacement of the VanA ω-loop by that of VanC2, a d-Ala-d-Ser ligase, eliminated d-Ala-d-lactate activity while improving by 3-fold the catalytic efficacy of d-Ala-d-Ala and d-Ala-d-Ser activity.