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 Achilles' heel of vancomycin-susceptible bacteria; binding of vancomycin to this sequence interferes with crosslinking and blocks cell-wall synthesis. A mutant enzyme (VanA) from vancomycin-resistant Enterococcus faecium has been found to incorporate alpha-hydroxy acids at the terminal site instead of D-Ala; the resulting depsipeptides do not bind vancomycin, yet function in the crosslinking reaction. To investigate the binding specificity of these ligases, we examined their inhibition by a series of substrate analogs.


RESULTS
Phosphinate and phosphonate dipeptide analogs (which, after phosphorylation by the enzyme, mimic intermediates in the ligation reaction) were prepared and evaluated as reversible inhibitors of the wild-type ligases DdlA and DdlB from Escherichia coli and of the mutant enzyme VanA. Ki values were calculated for the first stage of inhibitor binding according to a mechanism in which inhibitor competes with D-Ala for both substrate binding sites. DdlA is potently inhibited by phosphinates but not by phosphonates, while DdlB and VanA show little discrimination; both series of compounds inhibit 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 mechanism in which proton transfer from the attacking nucleophile to the departing phosphate occurs directly, without intervention of the enzyme.
Ellsworth
B A
BA
Department of Chemistry, University of California, Berkeley, CA 94720-1460, USA.
Tom
N J
NJ
Bartlett
P A
PA
D001419:Bacteria / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase