Engineered Amp C 尾-Lactamase as a Fluorescent Screening Tool for Class C 尾-Lactamase Inhibitors

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• 作者：Man-Wah Tsang ; Pak-Ho Chan ; Pui-Kin So ; Dik-Lung Ma ; Chun-Wai Tsang ; Kwok-Yin Wong ; Yun-Chung Leung
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：March 15, 2011
• 年：2011
• 卷：83
• 期：6
• 页码：1996-2004
• 全文大小：1059K
• 年卷期：v.83,no.6(March 15, 2011)
• ISSN：1520-6882

文摘

Class C 尾-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of 尾-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C 尾-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 尾-lactamase mutant designated as V211Cf for the in vitro screening of class C 尾-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme鈥檚 active site (position 211), Amp C 尾-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its 尾-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a 鈥渘atural鈥?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 尾-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C 尾-lactamases.