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-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) 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 (NH(2)) 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 omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). 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 omega-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 omega-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.
Lessard
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Healy
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Park
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Walsh
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D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism; D001120:Arginine / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D019729:Carbon-Oxygen Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D016983:Enterococcus / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism*; D007934:Leuconostoc / Q000201:enzymology; D008024:Ligands; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013237:Stereoisomerism; D013698:Templates, Genetic; D014640:Vancomycin / Q000737:chemistry*; D020713:Vancomycin Resistance
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004151:Dipeptides; D008024:Ligands; C071437:VanA ligase, Bacteria; C079914:VanC protein, Bacteria; C115095:acetyl-alanyl-lactate; D019344:Lactic Acid; D006639:Histidine; C033627:3,3-difluoroalanine; D014640:Vancomycin; D001120:Arginine; D000251:Adenosine Triphosphatases; D019729:Carbon-Oxygen Ligases; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase; D008239:Lysine; D000409:Alanine