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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 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  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.

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