Novel metagenome-derived carboxylesterase that hydrolyzes ?-lactam antibiotics.

ABSTRACT: It has been proposed that family VIII carboxylesterases and class C ?-lactamases are phylogenetically related; however, none of carboxylesterases has been reported to hydrolyze ?-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 ?-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 ?-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 ?-lactam antibiotics. EstU1 was able to hydrolyze first-generation ?-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 ?-lactam ring of antibiotics, implicating the predicted catalytic triad of EstU1 in both activities.

SUBMITTER: Jeon JH 

PROVIDER: S-EPMC3209169 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature