Article (Scientific journals)
The role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
Montagner, Caroline; Nigen, Michaël; Jacquin, Olivier et al.
2016In Journal of Biological Chemistry, 291 (31), p. 16124-16137
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Keywords :
β-lactamase; antibiotic resistance; circular dichroism (CD); enzyme catalysis; metalloenzyme; nuclear magnetic resonance (NMR); protein folding; zinc
Abstract :
[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.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Montagner, Caroline ;  Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Nigen, Michaël
Jacquin, Olivier
Willet, Nicolas ;  Université de Liège > Département de chimie (sciences) > Nanochimie et systèmes moléculaires
Dumoulin, Mireille  ;  Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines
Karsisiotis, Andreas Ioannis
Roberts, Gordon C. K.
Damblon, Christian ;  Université de Liège > Département de chimie (sciences) > Chimie biologique structurale
Redfield, Christina
Matagne, André  ;  Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines
Language :
English
Title :
The role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
Publication date :
2016
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
Volume :
291
Issue :
31
Pages :
16124-16137
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
Copyright (c) 2016, The American Society for Biochemistry and Molecular Biology.
Available on ORBi :
since 01 June 2016

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