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Publication Abstract from PubMed

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

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled beta-lactamase.,Wong WT, Au HW, Yap HK, Leung YC, Wong KY, Zhao Y BMC Struct Biol. 2011 Mar 28;11:15. doi: 10.1186/1472-6807-11-15. PMID:21443768^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.