BADAN-conjugated ß-lactamases as biosensors for ß-lactam antibiotic detection.

ß-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated ß-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for ß-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP ß-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to ß-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four ß-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 µM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated ß-lactamases as biosensing devices for ß-lactam antibiotics.

Thermostable ß-Lactamase Mutant with Its Active Site Conjugated with Fluorescein for Efficient ß-Lactam Antibiotic Detection.

Monitoring the ß-lactam antibiotic level has been an important task in food industry and clinical practice. Here, we report the development of a fluorescent PenP ß-lactamase, PenP-E166Cf/N170Q, for efficient ß-lactam antibiotic detection. It was constructed by covalently attaching fluorescein onto the active-site entrance of a thermostable E166Cf/N170Q mutant of a Bacillus licheniformis PenP ß-lactamase. It gave a fluorescence turn-on signal toward various ß-lactam antibiotics, where the fluorescence enhancement was attributed to the acyl-enzyme complex formed between PenP-E166Cf/N170Q and the ß-lactam antibiotic. It demonstrated enhanced signal stability over its parental PenP-E166Cf because of the suppressed hydrolytic activity by the N170Q mutation. Compared with our previously constructed PenPC-E166Cf biosensor, PenP-E166Cf/N170Q was more thermostable and advanced in detecting ß-lactams in terms of response time, signal stability, and detection limit. Positive fluorescence signals generated by E166Cf/N170Q in response to the penicillin-containing milk and mouse serum illustrated the feasibility of the biosensor for antibiotic detection in real samples. Taken together, our findings suggest the potential application of PenP-E166Cf/N170Q in biosensing ß-lactam antibiotics.

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

BACKGROUND: ß-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for ß-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 ß-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 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 ß-lactamase. CONCLUSIONS: Our structural studies indicate the biosensing mechanism of a fluorescein-labelled ß-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of ß-lactamase-based biosensor to detect the wide spectrum of ß-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 ß-lactamase inhibitors that target the Ω-loop and not the active site.