title: The Role of Active Site Flexible Loops in Catalysis and of Zinc in Conformational Stability of Bacillus cereus 569/H/9 β-Lactamase.
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 (ΔG(0)) 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 ΔG(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.
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and.
authors.forename: Caroline
authors.initials: C
authors.lastname: Montagner
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and.
authors.forename: Michaël
authors.initials: M
authors.lastname: Nigen
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and.
authors.forename: Olivier
authors.initials: O
authors.lastname: Jacquin
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Willet
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and.
authors.forename: Mireille
authors.initials: M
authors.lastname: Dumoulin
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom.
authors.forename: Andreas Ioannis
authors.initials: AI
authors.lastname: Karsisiotis
authors.affiliation: the Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, University of Leicester, Leicester LE1 9HN, United Kingdom, and.
authors.forename: Gordon C K
authors.initials: GC
authors.lastname: Roberts
authors.affiliation: Département de Chimie, Université de Liège, Institut de Chimie B6, 4000 Liège (Sart Tilman), Belgium.
authors.forename: Christian
authors.initials: C
authors.lastname: Damblon
authors.affiliation: the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Christina
authors.initials: C
authors.lastname: Redfield
authors.affiliation: From the Laboratoire d'Enzymologie et Repliement des Protéines, Centre d'Ingénierie des Protéines, and amatagne@ulg.ac.be.
authors.forename: André
authors.initials: A
authors.lastname: Matagne
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology*; D020134:Catalytic Domain; D057927:Hydrophobic and Hydrophilic Interactions; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D015032:Zinc / Q000737:chemistry*; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D001618:beta-Lactamases; D015032:Zinc
keywords: antibiotic resistance; circular dichroism (CD); enzyme catalysis; metalloenzyme; nuclear magnetic resonance (NMR); protein folding; zinc; β-lactamase