High Resolution Crystal Structures of the trans-Enamine Intermediates Formed by Sulbactam and Clavulanic Acid and E166A SHV-1 β-Lactamase

Pius S Padayatti,Marion S Helfand,Monica A Totir,Marianne P Carey,Paul R Carey,Robert A Bonomo,Focco Van Den Akker

doi:10.1074/jbc.m505333200

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.

Highlights

Bacterial infections are often treated using ␤-lactam antibiotics that inhibit the final stages of peptidoglycan synthesis, leading to bacterial cell death
We have presented here the high resolution crystal structures of the ␤-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 ␤-lactamase
If we make the reasonable assumption that the Raman scattering cross-sections are very similar for the three enamine species, since the enamine skeletons are all the same, we observe that the populations of the clavulanic acid and especially the sulbactam-based intermediates are considerably reduced when compared with the population produced by tazobactam

Summary

Introduction

Bacterial infections are often treated using ␤-lactam antibiotics that inhibit the final stages of peptidoglycan synthesis, leading to bacterial cell death. ␤-lactamases evolve to counteract the inhibitors by acquiring point mutations that result in resistance to inhibition, whereas the enzymes maintain sufficient activity to degrade ␤-lactam antibiotics [5, 6]. The initial step of the catalytic mechanism of inactivation of SHV-1 is ligand binding [18] followed by nucleophilic attack on the carbonyl carbon atom by the hydroxyl group of Ser-70, resulting in a covalent acyl intermediate and the opening of the ␤-lactam ring (Fig. 1B). Deacylation-deficient E166A ␤-lactamase mutants have been used to maximize the possibility of trapping its acyl-enzyme intermediates for crystallographic analysis [22,23,24] These important structures were obtained by soaking or co-crystallization experiments carried out in the absence of prior information of the type and amount of intermediate that is present within a crystal at any given time.

Methods

Results

Conclusion