The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase

Caroline Montagner (1) , Michael Nigen (1, 2) , Olivier Jacquin (1) , Nicolas Willet (1) , Mireille Dumoulin (1) , Andreas Ioannis Karsisiotis (3) , Gordon C. K. Roberts (4) , Christian Damblon (5) , Christina Redfield (6) , André Matagne (7)

1 Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines
2 UMR IATE - Ingénierie des Agro-polymères et Technologies Émergentes
3 School of Biological Sciences
4 Henry Wellcome Laboratories of Structural Biology
5 Département de Chimie
6 Department of Biochemistry
7 Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, Liege, Belgique

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus beta-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (Delta G(0)) of 32 +/- 2kJ.mol(-1). For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with Delta G(0) value of 65 +/- 1.4 kJ.mol(-1). These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity.

Domaines: Sciences du Vivant [q-bio]

Caroline Montagner, Michael Nigen, Olivier Jacquin, Nicolas Willet, Mireille Dumoulin, et al.. The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase. Journal of Biological Chemistry, 2016, 291 (31), pp.16124-16137. ⟨10.1074/jbc.M116.719005⟩. ⟨hal-01506509⟩