Structural and functional studies on class A β-lactamase-derived biosensors

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Structural and functional studies on class A β-lactamase-derived biosensors

 

Author: Wong, Wai-ting
Title: Structural and functional studies on class A β-lactamase-derived biosensors
Degree: Ph.D.
Year: 2011
Subject: Biosensors.
Beta lactamases.
Beta lactam antibiotics.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Applied Biology and Chemical Technology
Pages: xxi, 274 p. : ill. (some col.) ; 30 cm.
InnoPac Record: http://library.polyu.edu.hk/record=b2456205
URI: http://theses.lib.polyu.edu.hk/handle/200/6188
Abstract: The extensive use of beta-lactam antibiotics in veterinary and clinical applications has led to food contamination and reduced efficacy of β-lactam antibiotics as bacteria become antibiotic-resistant due to selective pressure. The major mechanism of antibiotic resistance in bacteria is β-lactamase that inactivates the β-lactam antibiotics. Recently, our group has constructed a "switch-on" fluorescent biosensor from a β-lactamase (Chan P.-H. et al.; J. Am Chem. Soc., 2004, 126, 4074) that can successfully detect trace amount of β-lactam antibiotics. In this project, the β-lactamase from Bacillus licheniformis 749/C (PenP) was engineered by site-directed mutagenesis to form the mutant E166C, in which the Glu166 residue in the Ω-loop is replaced with a cysteine residue. It was separately labeled with two thiol-reactive fluorophores, badan (b) and fluorescein-5-maleimide (f) to form two biosensors, PenP_E166Cb and PenP_E166Cf, respectively. This project concentrates on the study of the biosensing mechanisms of both biosensors in order to explain how they generate fluorescence changes upon β-lactam binding. The fluorescence studies of both PenP_E166Cb and PenP_E166Cf with various β-lactams (mainly cephalosporins) reveal that the formation of enzyme-substrate complex enhances the fluorescence of the biosensors significantly. For PenP_E166Cb, the response is selective, and only the addition of oxyimino-cephalosporins gave significant enhancement in fluorescence intensity. Structural studies of PenP_E166Cb and PenP_E166Cf, as well as their antibiotic-bound intermediates show that the biosensing mechanisms in E166Cb and E166Cf are different. The results show that the E166Cb only detects oxyimino-cephalosporins because of the steric clashes between the side chain of oxyimino-cephalosporins and the Ω-loop residues, which induces a change in the local environment around the fluorophore. But E166Cf detects all cephalosporins as the fluorescein label is likely to share a common space with the incoming β-lactam in the active site so that the fluorescein label is displaced from the active site to a more solvent exposed environment. Binding kinetics was studied by ESI-MS to investigate the binding rate of cefotaxime for PenP_E166Cb in comparison with the unlabeled enzyme E166C. We found that the flexibility of the Ω-loop in PenP_E166Cb is significantly increased and the Ω-loop changed to a new conformation after incorporation of the badan label. The new conformation results in the improved binding of cephalosporins with large side chains. Furthermore, based on the crystal structure of PenP_E166Cb, some residues that are in close proximity to the badan label were changed and the biosensing properties of the β-lactamase-based biosensor were improved.

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