Investigation of the mechanism and specificity of D -Ala -D -Ala ligases in bacterial cell wall biosynthesis and the VanA (D -Ala -D -Lac ligase) and VanC2 (D -Ala -D -Ser ligase) variants that underlie vancomycin resistance

Because of the threat of growing vancomycin resistance in enterococci and methicillin-resistant Staphylococcus aureus, the enzymes responsible for resistance are being studied in the hope of discovering ways to inhibit resistance, as well as, predict amino acid substitutions that could lead to increased vancomycin resistance. The VanC2 and VanA ligases found in vancomycin resistant bacteria synthesize D-Ala-D-Ser and D-Ala-D-Lac, respectively, which are incorporated into the termini of peptidoglycan (PG) intermediates and have a 10 to 1000-fold lower binding affinity for vancomycin compared to the normal D-Ala-D-Ala termini. Thus, vancomycin is less able to block the transpeptidation and transglycosylation of PG intermediates needed to form a stable cell wall.;The only available x-ray structure of a D-Ala-D-X ligase family member, the D-Ala-D-Ala ligase from E. coli (DdlB), was used as a basis for mutagenesis studies to determine the residues required for substrate specificity and catalysis by VanC2 and VanA. Mutagenesis of two active site residues in VanC2 eliminated D-Ala-D-Ser ligase activity but retained significant D-Ala-D-Ala activity, suggesting that these residues are responsible for recognition of D-Ser. Similarly, a residue in VanA was identified that might participate in both repelling the protonated (NH3+) form of D-Ala2 to minimize D-Ala-D-Ala synthesis and, conversely, binding D-Lac to promote synthesis of D-Ala-D-Lac.;Evidence was also obtained to further support the existence of Dalanyl phosphate (D-Ala-P) as an enzyme intermediate in the reactions catalyzed by D-Ala-D-X ligases. Positional isotope exchange experiments suggested that D-Ala-P could be formed in the enzyme active site of VanC2 and VanA prior to binding the second substrate, D-Ser or D-Lac, respectively. Presteady-state analysis of DldB and VanA detected a burst of ADP formation, implying that ADP and D-Ala-P rapidly accumulate on the active site and that a step later in catalysis, such as capture of D-Ala-P by the second substrate or product release, is significantly rate-limiting.;The knowledge gained about the reaction mechanism and determinants of substrate specificity could be combined with insights from the recently solved x-ray structure of VanA to design new antibiotics to be administered with vancomycin for treating infections caused by vancomycin resistant bacteria.