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

Man Wah Tsang, Pak Ho Chan, Pui Kin So, Dik Lung Ma, Chun Wai Tsang, Kwok Yin Wong, Yun Chung Leung

Research output: Journal article publicationJournal articleAcademic researchpeer-review

6 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)1996-2004
Number of pages9
JournalAnalytical Chemistry
Volume83
Issue number6
DOIs
Publication statusPublished - 15 Mar 2011

ASJC Scopus subject areas

  • Analytical Chemistry

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