Bacterial vancomycin resistance: Crystallographic structural analyses of D-Ala:D-Ala ligase

The tertiary structure of D-Ala:D-Ala ligase (DD-ligase) encoded by the ddlB gene of Escherichia coli, co-crystallized with S,R-methylphosphinate (phosphinate) and adenosine triphosphate (ATP), was determined using X-ray diffraction to 2.3 A resolution. A structural mechanism for enzyme catalysis and substrate specificity is proposed, in which a helix dipole and a hydrogen bond triad of Y216, S150 and E15 assist the binding of two D-alanine substrates. The notion of a \u27molecular filter\u27 of tyrosine is introduced, which determines the substrate linkage specificity. The \u27molecular filter\u27 Y216-OH prefers the binding of the second D-alanine by hydrogen bond interaction with its $\alpha$-NH$\sb3\sp+$ group while it disfavors D-lactate due to the repulsion of the two electronegative O atoms. Subsequently the structures of DD-ligase in complex with a D-Ala-D-$\alpha$-hydroxybutyrate (phosphonate) and ATP and a mutant ligase Y216F in complex with a phosphinate and ATP were determined to 2.2 A and 1.9 A resolution, respectively. From this structural information and a sequence comparison, it is proposed that a DD-ligase-like structure without a \u27molecular filter\u27 is responsible for the broadened specificity of D-Ala:D-Lac ligase (VanA) found in vancomycin-resistant strains of Enterococcus. A molecular mechanism for VanA catalysis is proposed and its tertiary structure is predicted.