title: Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled β-lactamase.
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 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.
authors: Department of Applied Biology and Chemical Technology, Central Laboratory of Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Hong Hong, China.
authors: Wai-Ting
authors: WT
authors: Wong
authors: Ho-Wah
authors: HW
authors: Au
authors: Hong-Kin
authors: HK
authors: Yap
authors: Yun-Chung
authors: YC
authors: Leung
authors: Kwok-Yin
authors: KY
authors: Wong
authors: Yanxiang
authors: Y
authors: Zhao
mesh_terms: D000900:Anti-Bacterial Agents / Q000032:analysis*; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D015374:Biosensing Techniques / Q000379:methods*; D020134:Catalytic Domain; D002439:Cefotaxime / Q000378:metabolism; D055808:Drug Discovery; D019793:Fluorescein / Q000737:chemistry*; D008958:Models, Molecular; D015203:Reproducibility of Results; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D001051:Apoenzymes; D001618:beta-Lactamases; D002439:Cefotaxime; D019793:Fluorescein