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Please use this identifier to cite or link to this item: http://hdl.handle.net/10397/5114

Title: Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase
Authors: Wong, Wai-ting
Au, Ho-wah
Yap, Hongkin
Leung, Thomas Y. C.
Wong, Kwok-yin
Zhao, Y. X.
Subjects: Cephalosporins
Beta lactam antibiotics
Anti-infective agents
Radioactivity
Biosensors
Issue Date: 28-Mar-2011
Publisher: BioMed Central Ltd.
Citation: BMC structural biology, 2011, v. 11, 15, p. 1-8.
Abstract: Background: β-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood.
Results: Here we report the crystal structure of a Class A β-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf) and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime), to illustrate its biosensing mechanism. In the apo structure the fluorescein molecule partially occupies the antibiotic binding site and is highly dynamic. In the PenPE166Cf-cefatoxime complex structure the binding and subsequent acylation of cefotaxime to PenP displaces fluorescein from its original location to avoid steric clash. Such displacement causes the well-folded Ω-loop to become fully flexible and the conjugated fluorescein molecule to relocate to a more solvent exposed environment, hence enhancing its fluorescence emission. Furthermore, the fully flexible Ω-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum β-lactamase.
Conclusions: Our structural studies indicate the biosensing mechanism of a fluorescein-labelled β-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of β-lactamase-based biosensor to detect the wide spectrum of β-lactam antibiotics. The observation of increased Ω-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel β-lactamase inhibitors that target the Ω-loop and not the active site.
Description: DOI: 10.1186/1472-6807-11-15
Rights: © 2011 Wong et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Type: Journal/Magazine Article
URI: http://hdl.handle.net/10397/5114
ISSN: 1472-6807
Appears in Collections:ABCT Journal/Magazine Articles

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