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

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Abstract
Escherichia colid-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 ω-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 Gram-positive bacteria with intrinsic resistance to vancomycin.

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