Download MendeleyHigh Resolution Crystal Structures of the trans-Enamine Intermediates Formed by Sulbactam and Clavulanic Acid and E166A SHV-1 {beta}-Lactamase.
Padayatti, P.S., Helfand, M.S., Totir, M.A., Carey, M.P., Carey, P.R., Bonomo, R.A., van den Akker, F.(2005) J Biological Chem 280: 34900-34907
- PubMed: 16055923
- DOI: https://doi.org/10.1074/jbc.M505333200
- Primary Citation of Related Structures:
2A3U, 2A49 - PubMed Abstract:
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.
Organizational Affiliation:
Department of Biochemistry, Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
