title: Engineered Amp C β-lactamase as a fluorescent screening tool for class C β-lactamase inhibitors.
abstract: 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.
authors.affiliation: Department of Applied Biology and Chemical Technology, Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
authors.forename: Man-Wah
authors.initials: MW
authors.lastname: Tsang
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.forename: Pui-Kin
authors.initials: PK
authors.lastname: So
authors.forename: Dik-Lung
authors.initials: DL
authors.lastname: Ma
authors.forename: Chun-Wai
authors.initials: CW
authors.lastname: Tsang
authors.forename: Kwok-Yin
authors.initials: KY
authors.lastname: Wong
authors.forename: Yun-Chung
authors.initials: YC
authors.lastname: Leung
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D016972:Enterobacter cloacae / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D013050:Spectrometry, Fluorescence; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; C048795:AmpC beta-lactamases; D001618:beta-Lactamases