High resolution crystal structures of the trans-enamine intermediates formed by sulbactam and clavulanic acid and E166A SHV-1 {beta}-lactamase

• Published in: The Journal of biological chemistry 2005-10, Vol.280 (41), p.34900-34907
• Main Authors: Padayatti, Pius S, Helfand, Marion S, Totir, Monica A, Carey, Marianne P, Carey, Paul R, Bonomo, Robert A, van den Akker, Focco
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; beta-Lactam Resistance; beta-Lactamases - chemistry; Binding Sites; Clavulanic Acid - pharmacology; Crystallography, X-Ray; Drug Resistance, Bacterial; Enzyme Inhibitors - pharmacology; Klebsiella pneumoniae - metabolism; Models, Chemical; Models, Molecular; Mutation; Penicillanic Acid - analogs & derivatives; Penicillanic Acid - pharmacology; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Serine - chemistry; Spectrum Analysis, Raman; Sulbactam - pharmacology; Tazobactam; Time Factors; X-Rays
• ISSN: 0021-9258

Antibiotic resistance mediated by constantly evolving beta-lactamases is a serious threat to human health. The mechanism of inhibition of these enzymes by therapeutic beta-lactamase inhibitors is probed using a novel approach involving Raman microscopy and x-ray crystallography. We have presented here the high resolution crystal structures of the beta-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 beta-lactamase. Our previous Raman measurements have identified the trans-enamine species for both inhibitors and were used to guide the soaking time and concentration to achieve full occupancy of the active sites. The two inhibitor-bound x-ray structures revealed a linear trans-enamine intermediate covalently attached to the active site Ser-70 residue. This intermediate was thought to play a key role in the transient inhibition of class A beta-lactamases. Both the Raman and x-ray data indicated that the clavulanic acid intermediate is decarboxylated. When compared with our previously determined tazobactam-bound inhibitor structure, our new inhibitor-bound structures revealed an increased disorder in the tail region of the inhibitors as well as in the enamine skeleton. The x-ray crystallographic observations correlated with the broadening of the O-C=C-N (enamine) symmetric stretch Raman band near 1595 cm(-1). Band broadening in the sulbactam and clavulanic acid inter-mediates reflected a heterogeneous conformational population that results from variations of torsional angles in the O-(C=O)-C=C=NH-C skeleton. These observations led us to conclude that the conformational stability of the trans-enamine form is critical for their transient inhibitory efficacy.