D-ALANINE-D-ALANINE LIGASE - PHOSPHONATE AND PHOSPHINATE INTERMEDIATES WITH TYPE AND THE Y216F MUTANT

The crystallographic structure of the D-alanine:D-alanine ligase of th e ddlB gene of Escherichia coli complexed with a D-Ala-D-a-hydroxybuty rate phosphonate and the structure of the Y216F mutant ligase complexe d with a D-Ala-D-Ala phosphinate have been determined to 2.2 and 1.9 A ngstrom resolution, respectively, and refined to R factors of 0.156 an d 0.158. In each complex the inhibitor has reacted with ATP to produce ADP and a tight-binding phosphorylated transition state intermediate. Comparison of these two structures with the known crystal structure o f the phosphinate intermediate of the wild-type ligase shows no major conformational changes, but B factors indicate differences in mobility of loops covering the binding site. The weaker inhibition of the Y216 F mutant by both inhibitors is thought to be due in part to the loss o f an interloop hydrogen bond. A similar mechanism may account for poor inhibition of VanA, the homologous D-Ala:D-lactate ligase produced by vancomycin-resistant enterococci.