Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-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 firstgeneration ␤-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.

Challenging the concept of bacteria subsisting on antibiotics

“…Staphylococcus aureus ATCC Cultures of CA-S3F-1, PE-C-1 and PE-S2R-1 grown in the respective antibiotic (1 mg/mL)-containing LB broth at 26 • C with shaking overnight were diluted 1 in 10 in antibiotic (1 mg/mL)-containing LB broth and incubated for a further 4 h. The filtered (0.2 m filters), washed supernatants were used in the crude ␤-lactamase enzyme assays using nitrocefin at 100 M at a wavelength of 486 nm [16]. Escherichia coli transformed with pUC19 plasmid (AmpC ␤-lactamase-producing) was used as a positive control.…”

Discovery of Polyesterases from Moss-Associated Microorganisms

“…The K m values determined for EstB3 and EstC7 were very low, suggesting high affinity for pNPB as a substrate. For comparison, K m values for other family VIII carboxylesterases from metagenome sources, such as those for EstC (58.7 M) (47) and EstU1 (6.0 M) (40), are in a similar range. The recently described esterase Cbotu_EstA, which is also capable of hydrolyzing PBAT, has a V max value similar to that of EstC7 (Cbotu_EstA V max ϭ 83.4 U mg Ϫ1 ); however, the K m value for Cbotu_EstA is 3 orders of magnitude higher (1.95 mM) (20).…”

“…4IVK) (39). Promiscuous ␤-lactamase activity has been demonstrated for some members of this family, such as the metagenome-derived carboxylesterases EstU1 (40) and Est22 (41), with first-generation cephalosporin-based derivatives. EstG4 clustered in family II of lipolytic enzymes, which show the conserved motif GDSL containing the active site serine.…”

An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range

“…As a result, family VIII esterases are typically tested for their ability to hydrolyse a variety of β-lactam substrates ( Table 2). The majority either lack the activity or show negligible activity, despite the high sequence identity to the β-lactamases, while others have been described as exhibiting "promiscuous β-lactamase activity" [18,21,41,47,48,49]. Consequently, it has been suggested that these esterases have evolved from the class C β-lactamases, where some have maintained this remnant activity, while others have lost the capability due to steric interference resulting from structural evolution [18,41,50].…”

“…Interestingly, detailed selectivity assays conducted for Est22 [49] and EstB [40] demonstrated that the enzymes selectively hydrolysed the ester bond of a number of cephalosporin-based substrates, while leaving the amide bond of the β-lactam ring intact. This clearly differentiates them from bona fide β-lactamases.…”