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.