University of Leicester
J. Biol. Chem.-2016-Montagner-16124-37.pdf (2.47 MB)

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

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posted on 2017-05-15, 11:30 authored by Caroline Montagner, Michaël Nigen, Olivier Jacquin, Nicolas Willet, Mireille Dumoulin, Andreas Ioannis Karsisiotis, Gordon C. K. Roberts, Christian Damblon, Christina Redfield, André Matagne
Metallo-β-lactamases catalyze the hydrolysis of most β-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 β-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 (ΔG0) of 32 ± 2 kJ·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 ΔG0 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.



Journal of Biological Chemistry, 2016, 291 (31), pp. 16124-16137

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/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Old Departments Pre 01 Aug 2015/Department of Biochemistry (Pre 01 Aug 2015)


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