Engineered Amp C β-lactamase as a fluorescent screening tool for class C β-lactamase inhibitors

Anal Chem. 2011 Mar 15;83(6):1996-2004. doi: 10.1021/ac102595r. Epub 2011 Feb 21.


Class C β-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of β-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C β-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C β-lactamase mutant designated as V211Cf for the in vitro screening of class C β-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C β-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its β-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C β-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C β-lactamases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / metabolism
  • Anti-Bacterial Agents / pharmacology
  • Bacterial Proteins / antagonists & inhibitors*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Biocatalysis
  • Drug Evaluation, Preclinical / methods*
  • Enterobacter cloacae / enzymology
  • Enzyme Inhibitors / metabolism
  • Enzyme Inhibitors / pharmacology*
  • Models, Molecular
  • Mutation
  • Protein Conformation
  • Protein Engineering*
  • Spectrometry, Fluorescence
  • beta-Lactamase Inhibitors*
  • beta-Lactamases / chemistry
  • beta-Lactamases / genetics*
  • beta-Lactamases / metabolism
  • beta-Lactams / metabolism
  • beta-Lactams / pharmacology


  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Enzyme Inhibitors
  • beta-Lactamase Inhibitors
  • beta-Lactams
  • AmpC beta-lactamases
  • beta-Lactamases