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

BMC Struct Biol. 2011 Mar 28;11:15. doi: 10.1186/1472-6807-11-15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / analysis*
  • Apoenzymes / chemistry
  • Apoenzymes / metabolism
  • Bacillus / enzymology
  • Biosensing Techniques / methods*
  • Catalytic Domain
  • Cefotaxime / metabolism
  • Drug Discovery
  • Fluorescein / chemistry*
  • Models, Molecular
  • Reproducibility of Results
  • beta-Lactamases / chemistry*
  • beta-Lactamases / metabolism*

Substances

  • Anti-Bacterial Agents
  • Apoenzymes
  • beta-Lactamases
  • Cefotaxime
  • Fluorescein