title: Novel metagenome-derived carboxylesterase that hydrolyzes β-lactam antibiotics.
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.
authors: Marine Biotechnology Research Center, Korea Ocean Research and Development Institute, Ansan, South Korea.
authors: Jeong Ho
authors: JH
authors: Jeon
authors: Soo-Jin
authors: SJ
authors: Kim
authors: Hyun Sook
authors: HS
authors: Lee
authors: Sun-Shin
authors: SS
authors: Cha
authors: Jung Hun
authors: JH
authors: Lee
authors: Sang-Hong
authors: SH
authors: Yoon
authors: Bon-Sung
authors: BS
authors: Koo
authors: Chang-Muk
authors: CM
authors: Lee
authors: Sang Ho
authors: SH
authors: Choi
authors: Sang Hee
authors: SH
authors: Lee
authors: Sung Gyun
authors: SG
authors: Kang
authors: Jung-Hyun
authors: JH
authors: Lee
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D043182:Carboxylesterase / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D006868:Hydrolysis; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013379:Substrate Specificity; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D050505:Mutant Proteins; D011994:Recombinant Proteins; D047090:beta-Lactams; D043182:Carboxylesterase