1IEM : Crystal Structure of AmpC beta-lactamase from E. coli in Complex with a Boronic Acid Inhibitor (1, CefB4)

  • Fabio Prati (Contributor)
  • R. A. Powers (Contributor)
  • Emilia Caselli (Contributor)
  • Pamela J Focia (Contributor)
  • Brian K. Shoichet (Contributor)



Experimental Technique/Method:X-RAY DIFFRACTION
Release Date:2001-08-15
Deposition Date:2001-04-10
Revision Date:2008-04-27#2011-07-13
Molecular Weight:79931.06
Macromolecule Type:Protein
Residue Count:716
Atom Site Count:5613

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.
Date made available2001

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