Novel Metagenome-Derived Carboxylesterase That Hydrolyzes beta-Lactam Antibiotics
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Title
- Novel Metagenome-Derived Carboxylesterase That Hydrolyzes beta-Lactam Antibiotics
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Author(s)
- Jeon, Jeong Ho; Kim, Soo-Jin; Lee, Hyun Sook; Cha, Sun-Shin; Lee, Jung Hun; Yoon, Sang-Hong; Koo, Bon-Sung; Lee, Chang-Muk; Choi, Sang Ho; Lee, Sang Hee; Kang, Sung Gyun; Lee, Jung-Hyun
- KIOST Author(s)
- Lee, Hyun Sook(이현숙); Kang, Sung Gyun(강성균); Lee, Jung Hyun(이정현)
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Alternative Author(s)
- 전정호; 이현숙; 차선신; 강성균; 이정현
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Publication Year
- 2011-11
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Abstract
- It has been proposed that family VIII carboxylesterases and class C beta-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze beta-lactam antibiotics except nitrocefin, a nonclinical chromogenic substrate. Here, we describe the first example of a novel carboxylesterase derived from a metagenome that is able to cleave the amide bond of various beta-lactam substrates and the ester bond of p-nitrophenyl esters. A clone with lipolytic activity was selected by functional screening of a metagenomic library using tributyrin agar plates. The sequence analysis of the clone revealed the presence of an open reading frame (estU1) encoding a polypeptide of 426 amino acids, retaining an S-X-X-K motif that is conserved in class C beta-lactamases and family VIII carboxylesterases. The gene was overexpressed in Escherichia coli, and the purified recombinant protein (EstU1) was further characterized. EstU1 showed esterase activity toward various chromogenic p-nitrophenyl esters. In addition, it exhibited hydrolytic activity toward nitrocefin, leading us to investigate whether EstU1 could hydrolyze beta-lactam antibiotics. EstU1 was able to hydrolyze first-generation beta-lactam antibiotics, such as cephalosporins, cephaloridine, cephalothin, and cefazolin. In a kinetic study, EstU1 showed a similar range of substrate affinities for both p-nitrophenyl butyrate and first-generation cephalosporins while the turnover efficiency for the latter was much lower. Furthermore, site-directed mutagenesis studies revealed that the catalytic triad of EstU1 plays a crucial role in hydrolyzing both ester bonds of p-nitrophenyl esters and amide bonds of the beta-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.
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ISSN
- 0099-2240
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URI
- https://sciwatch.kiost.ac.kr/handle/2020.kiost/3797
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DOI
- 10.1128/AEM.05363-11
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Bibliographic Citation
- APPLIED AND ENVIRONMENTAL MICROBIOLOGY, v.77, no.21, pp.7830 - 7836, 2011
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Publisher
- AMER SOC MICROBIOLOGY
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Subject
- LIPASES; MECHANISM; ENZYMES; FAMILY; CLASSIFICATION; BIOTECHNOLOGY; RESISTANCE; HYDROLASE; EVOLUTION; SUBSTRATE
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Type
- Article
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Language
- English
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Document Type
- Article
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