The structures of Ceftazidime and Its Transition State Analog in Complex with AmpC-beta-lactamase: Implication for Resistance Mutation and Inhibitor Design

Third-generation cephalosporins are widely used ‚-lactam antibiotics that resist hydrolysis by‚-lactamases. Recently, mutant ‚-lactamases that rapidly inactivate these drugs have emerged. To investigatewhy third-generation cephalosporins are relatively stable to wild-type class C ‚-lactamases and how mutantenzymes might overcome this, the structures of the class C ‚-lactamase AmpC in complex with the thirdgenerationcephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determinedby X-ray crystallography to 2.0 and 2.3 Å resolution, respectively. Comparison of the acyl-enzyme structuresof ceftazidime and loracarbef, a ‚-lactam substrate, reveals that the conformation of ceftazidime in theactive site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediateand the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transitionstate, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent forcatalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-raycrystal structure of the mutant ‚-lactamase GC1, which has improved activity against third-generationcephalosporins, suggests that a tandem tripeptide insertion in the ø loop, which contains Val211, hascaused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generationcephalosporins to adopt a more catalytically competent conformation. These structural differences mayexplain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, thecomplexed structure of the transition-state analogue inhibitor (Ki 20 nM) with AmpC reveals potentialopportunities for further inhibitor design.