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Title: The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase
Authors: Montagner, Caroline
Nigen, Michaël
Jacquin, Olivier
Willet, Nicolas
Dumoulin, Mireille
Karsisiotis, Andreas Ioannis
Roberts, Gordon C. K.
Damblon, Christian
Redfield, Christina
Matagne, André
First Published: 27-May-2016
Publisher: American Society for Biochemistry and Molecular Biology
Citation: Journal of Biological Chemistry, 2016, 291 (31), pp. 16124-16137
Abstract: 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.
DOI Link: 10.1074/jbc.M116.719005
ISSN: 0021-9258
eISSN: 1083-351X
Version: Publisher Version
Status: Peer-reviewed
Type: Journal Article
Rights: Copyright © 2016 The American Society for Biochemistry and Molecular Biology, Inc. Deposited with reference to the publisher’s open access archiving policy.
Appears in Collections:Published Articles, Dept. of Biochemistry

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