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

Journal Article · Mon Mar 08 04:00:00 EST 2010 · Biochemistry-US

OSTI ID:1006361

Powers, R A; Caselli, E; Focia, P J; Prati, F; Shoichet, B K

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 {angstrom} 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{sub i} 20 nM) with AmpC reveals potential opportunities for further inhibitor design.

• Research Organization: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)
• Sponsoring Organization: USDOE
• OSTI ID: 1006361
• Journal Information: Biochemistry-US, Journal Name: Biochemistry-US Journal Issue: (31) ; 08, 2001 Vol. 40; ISSN 0006-2960; ISSN BICHAW
• Country of Publication: United States
• Language: ENGLISH

Related Subjects

36 MATERIALS SCIENCE
ANTIBIOTICS
CATALYSIS
CRYSTAL STRUCTURE
CRYSTALLOGRAPHY
DESIGN
ENZYMES
HYDROLYSIS
MUTANTS
MUTATIONS
RESIDUES
RESOLUTION
SUBSTRATES