High resolution crystal structures of the trans-enamine intermediates formed by sulbactam and clavulanic acid and E166A SHV-1 {beta}-lactamase.
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.
Padayatti
Pius S
PS
Department of Biochemistry, Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Helfand
Marion S
MS
Totir
Monica A
MA
Carey
Marianne P
MP
Carey
Paul R
PR
Bonomo
Robert A
RA
van den Akker
Focco
F
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D019818:Clavulanic Acid / Q000494:pharmacology*; D018360:Crystallography, X-Ray; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007711:Klebsiella pneumoniae / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D010397:Penicillanic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D013407:Sulbactam / Q000494:pharmacology*; D000078142:Tazobactam; D013997:Time Factors; D014965:X-Rays; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000737:chemistry*
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D019818:Clavulanic Acid; D012694:Serine; D010397:Penicillanic Acid; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases; D013407:Sulbactam; D000078142:Tazobactam