Reference : The role of active site flexible loops in catalysis and of zinc in conformational sta...
Scientific journals : Article
Life sciences : Biochemistry, biophysics & molecular biology
http://hdl.handle.net/2268/197661
The role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
English
Montagner, Caroline mailto [Université de Liège > Département des sciences de la vie > Macromolécules biologiques >]
Nigen, Michaël [> >]
Jacquin, Olivier [> >]
Willet, Nicolas mailto [Université de Liège > Département de chimie (sciences) > Nanochimie et systèmes moléculaires >]
Dumoulin, Mireille mailto [Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines >]
Karsisiotis, Andreas Ioannis [> >]
Roberts, Gordon C. K. [> >]
Damblon, Christian mailto [Université de Liège > Département de chimie (sciences) > Chimie biologique structurale >]
Redfield, Christina [> >]
Matagne, André mailto [Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines >]
2016
Journal of Biological Chemistry
American Society for Biochemistry and Molecular Biology
291
31
16124-16137
Yes (verified by ORBi)
International
0021-9258
1083-351X
Baltimore
MD
[en] β-lactamase ; antibiotic resistance ; circular dichroism (CD) ; enzyme catalysis ; metalloenzyme ; nuclear magnetic resonance (NMR) ; protein folding ; zinc
[en] Metallo-beta-lactamases catalyse 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 (DeltaG degrees ) of 32 +/- 2 kJ.mol11. 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. 2D 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 DeltaG degrees value of 65 +/- 1.4 kJ.mol11. 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 in order to maintain enzymatic activity.
http://hdl.handle.net/2268/197661
10.1074/jbc.M116.719005
Copyright (c) 2016, The American Society for Biochemistry and Molecular Biology.

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