NDLI: Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics ▿ †

Content Provider	PubMed Central
Author	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
Copyright Year	2011
Abstract	It has been proposed that family VIII carboxylesterases and class C β-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze β-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 β-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 β-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 β-lactam antibiotics. EstU1 was able to hydrolyze first-generation β-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 β-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.
Related Links	http://dx.doi.org/10.1128/aem.05363-11
Ending Page	7836
Page Count	7
Starting Page	7830
File Format	PDF
ISSN	00992240
e-ISSN	10985336
Journal	Applied and Environmental Microbiology
Issue Number	21
Volume Number	77
Language	English
Publisher	American Society for Microbiology
Publisher Date	2011-11-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Biotechnology Food Science Ecology Applied Microbiology and Biotechnology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Ecology Food Science Applied Microbiology and Biotechnology Biotechnology

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