Structures of ceftazidime and its transition-state analogue in complex with AmpC beta-lactamase: implications for resistance mutations and inhibitor design

Biochemistry. 2001 Aug 7;40(31):9207-14. doi: 10.1021/bi0109358.


Third-generation cephalosporins are widely used beta-lactam antibiotics that resist hydrolysis by beta-lactamases. Recently, mutant beta-lactamases that rapidly inactivate these drugs have emerged. To investigate why third-generation cephalosporins are relatively stable to wild-type class C beta-lactamases and how mutant enzymes might overcome this, the structures of the class C beta-lactamase AmpC in complex with the third-generation cephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determined by X-ray crystallography to 2.0 and 2.3 A resolution, respectively. Comparison of the acyl-enzyme structures of ceftazidime and loracarbef, a beta-lactam substrate, reveals that the conformation of ceftazidime in the active site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediate and the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transition state, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent for catalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-ray crystal structure of the mutant beta-lactamase GC1, which has improved activity against third-generation cephalosporins, suggests that a tandem tripeptide insertion in the Omega loop, which contains Val211, has caused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generation cephalosporins to adopt a more catalytically competent conformation. These structural differences may explain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, the complexed structure of the transition-state analogue inhibitor (K(i) 20 nM) with AmpC reveals potential opportunities for further inhibitor design.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins*
  • Boronic Acids / chemistry
  • Ceftazidime / analogs & derivatives*
  • Ceftazidime / chemistry*
  • Cephalosporins / chemistry
  • Crystallography, X-Ray
  • Drug Resistance, Microbial / genetics
  • Enzyme Inhibitors / chemical synthesis
  • Enzyme Inhibitors / chemistry*
  • Escherichia coli / enzymology
  • Macromolecular Substances
  • Mutagenesis, Site-Directed
  • beta-Lactamase Inhibitors*
  • beta-Lactamases / chemistry*
  • beta-Lactamases / genetics


  • Bacterial Proteins
  • Boronic Acids
  • Cephalosporins
  • Enzyme Inhibitors
  • Macromolecular Substances
  • beta-Lactamase Inhibitors
  • Ceftazidime
  • AmpC beta-lactamases
  • beta-Lactamases

Associated data

  • PDB/1IEL
  • PDB/1IEM