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Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled ?-lactamase.

ABSTRACT: BACKGROUND: ?-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the ?-loop near its catalytic site is a highly effective biosensor for ?-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 ?-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 PenP-E166Cf-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 ?-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 ?-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum ?-lactamase. CONCLUSIONS: Our structural studies indicate the biosensing mechanism of a fluorescein-labelled ?-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of ?-lactamase-based biosensor to detect the wide spectrum of ?-lactam antibiotics. The observation of increased ?-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel ?-lactamase inhibitors that target the ?-loop and not the active site.

SUBMITTER: Wong WT

PROVIDER: S-EPMC3076226 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase.

Wong Wai-Ting WT Au Ho-Wah HW Yap Hong-Kin HK Leung Yun-Chung YC Wong Kwok-Yin KY Zhao Yanxiang Y

BMC structural biology 20110328

<h4>Background</h4>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.<h4>Results</h4>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-E166C ...[more]

PMID: 21443768

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Project description:Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Dataset Information

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled ?-lactamase.

Publications

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase.

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