Novel Metagenome-Derived Carboxylesterase That Hydrolyzes [Beta]-Lactam Antibiotics

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...

Full description

Saved in:
Bibliographic Details
Published in:Applied and environmental microbiology 2011-11, Vol.77 (21), p.7830
Main Authors: 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
Format: Article
Language:English
Subjects:
Antibiotics
Chemical bonds
E coli
Gene expression
Microbiology
Proteins
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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. [PUBLICATION ABSTRACT]
ISSN:0099-2240
1098-5336