SYNTHESIS AND EVALUATION OF INHIBITORS OF BACTERIAL D-ALANINE-D-ALANINE LIGASES

Background: D-Alanine:D-alanine Ligase is essential for bacterial cell wall synthesis, assembling one of the subunits used for peptidoglycan crosslinking. The resulting aminoacyl-D-Ala-D-Ala strand is the Achil les' heel of vancomycin-susceptible bacteria, binding of vancomycin to this sequence interferes with crosslinking and blocks cell-wall synth esis. A mutant enzyme (VanA) from vancomycin-resistant Enterococcus fa ecium has been found to incorporate oc-hydroxy acids at the terminal s ite instead of D-Ala; the resulting depsipeptides do not bind vancomyc in, yet function in the crosslinking reaction. To investigate the bind ing specificity of these ligases, we examined their inhibition by a se ries of substrate analogs. Results: Phosphinate and phosphonate dipept ide analogs (which, after phosphorylation by the enzyme, mimic interme diates in the ligation reaction) were prepared and evaluated as revers ible inhibitors of the wild-type ligases DdlA and DdlB from Escherichi a coli and of the mutant enzyme VanA. Ki values were calculated for th e first stage of inhibitor binding according to a mechanism in which i nhibitor competes with D-Ala for both substrate binding sites. DdlA is potently inhibited by phosphinates but not by phosphonates, while Ddl B and VanA show little discrimination; both series of compounds inhibi t DdlB strongly and VanA weakly. Conclusions: VanA has greatly reduced affinity for all the Ligands studied. The relative affinities of the inhibitors in the reversible binding step are not, however, consistent with the substrate specificities of the enzymes. We propose a mechani sm in which proton transfer from the attacking nucleophile to the depa rting phosphate occurs directly, without intervention of the enzyme.