Gain of D-alanyl-D-lactate or D-lactyl-D-alanine synthetase activities in three active-site mutants of the Escherichia coli D-alanyl-D-alanine ligase B.
Escherichia coli D-Ala-D-Ala ligase (Ddl) and the vancomycin resistance-conferring protein VanA are homologues, but VanA has gained the ability to activate D-lactate (D-Lac) and make the depsipeptide D-Ala-D-Lac as well as D-Ala-D-Ala. This depsipeptide ligase activity of VanA is its crucial catalytic function necessary for phenotypic vancomycin resistance. We report here that three E. coli DdlB active-site mutants that we made previously based on X-ray structure/function predictions have gained interesting new ligase activities. Y216, S150, and E15 form a hydrogen-bonding triad that orients an omega-loop to close over the active site and also to orient substrate D-Ala1. Mutants Y216F and S150A have gained depsipeptide (D-Ala-D-Lac, D-Ala-D-hydroxybutyrate) ligase activity with dipeptide/depsipeptide partition ratios that mimic the pH behavior of VanA. E15Q has negligible depsipeptide synthetase activity but now uniquely activates D-Lac as the electrophilic rather than the nucleophilic partner for condensation with D-Ala to make a regioisomeric D-Lac-D-Ala, an amide rather than an ester product. These results provide insights into the active-site architecture of the ligases and the subsites for recognition of D-Ala VS D-Lac and predict the Y216F substitution will impart D-Ala-D-Lac synthetase activity to Ddls from Grampositive bacteria with intrinsic resistance to vancomycin.
Park
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lin
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Walsh
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D000409:Alanine / Q000031:analogs & derivatives* / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D019729:Carbon-Oxygen Ligases; D004351:Drug Resistance; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007773:Lactates / Q000378:metabolism*; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010453:Peptide Synthases / Q000378:metabolism*; D013379:Substrate Specificity; D014640:Vancomycin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D007773:Lactates; C071437:VanA ligase, Bacteria; C078877:alanyllactate; D014640:Vancomycin; D008025:Ligases; D019729:Carbon-Oxygen Ligases; D010453:Peptide Synthases; C101079:lactylalanine synthetase; C019638:D-alanylalanine synthetase; D000409:Alanine