Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Yu, Eun Young; Kwon, Min-A; Lee, Mi-Ae; Oh, Joon Young; Choi, Jongmin; Lee, Jun-Young; Song, Bongkeun; Hahm, Dae‐Hyun; Song, Jae Kwang

doi:10.1007/s00253-011-3132-7

Cited by 72 publications

(43 citation statements)

References 27 publications

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“…The discovery and characterization of carboxylesterases, including family VIII, have previously been reported in other metagenomic studies (12,19,26,33). A relationship between family VIII carboxylesterases and ␤-lactamase has often been proposed due to the presence of the S-X-X-K motif; however, no single enzyme has been demonstrated to have both esterase and ␤-lactamase activities.…”

Section: Discussionmentioning

confidence: 99%

“…A relationship between family VIII carboxylesterases and ␤-lactamase has often been proposed due to the presence of the S-X-X-K motif; however, no single enzyme has been demonstrated to have both esterase and ␤-lactamase activities. Recently, it has been reported that proteins of metagenomic origin belonging to family VIII carboxylesterases (26,33) could hydrolyze nitrocefin, which is a chromogenic substrate for assessing ␤-lactamase activity. However, none of these enzymes was able to hydrolyze ␤-lactam antibiotic substrates such as ampicillin, carbenicillin, cephalosporin C, or cephalothin.…”

Section: Discussionmentioning

confidence: 99%

“…In recent studies, family VIII carboxylesterases have been identified from metagenomic libraries (12,19,26,33). Among these proteins, EstC, derived from a leachate metagenome library, and two carboxylesterases, EstM-N1 and EstM-N2, derived from an arctic soil metagenome library, displayed a notable catalytic feature: in addition to their carboxylesterase activity, they also exhibited hydrolyzing activity toward nitrocefin, a chromogenic substrate used to determine ␤-lactamase activity (26,33). However, none of the carboxylesterases, including EstC, EstM-N1, and EstM-N2, has shown hydrolyzing activity toward ␤-lactam antibiotics.…”

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confidence: 99%

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Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

Jeon

Kim

Lee

et al. 2011

Appl Environ Microbiol

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

Jeon

Kim

Lee

et al. 2011

Appl Environ Microbiol

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“…The closest characterised relative was EstM-N2 (49% amino acid identity), a metagenome derived cold-active esterase belonging to the family VIII esterase/lipase and class C β-lactamases [37]. Further sequence A c c e p t e d M a n u s c i analysis searches using the Lipase Engineering database [38] and the Arpigny and Jaeger classification scheme [5] indicated that EstG34 was a family VIII carboxylesterase.…”

Section: Resultsmentioning

confidence: 99%

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

Ohlhoff

Kirby

Zyl

et al. 2015

Journal of Molecular Catalysis B: Enzymatic

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“…3). 41,42 This group contains the Class C b-lactamases and a number of b-lactamase like proteins, though the exact function of many of these proteins has yet to be biochemically determined.…”

Section: Analysis Of the Abgt Genementioning

confidence: 99%

Inhibition of the growth ofBacillus subtilisDSM10 by a newly discovered antibacterial protein from the soil metagenome

et al. 2015

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(2015) Inhibition of the growth of Bacillussubtilis DSM10 by a newly discovered antibacterial protein from the soil metagenome, Bioengineered, 6:2, 89-98, DOI: 10.1080/21655979.2015 A functional metagenomics based approach exploiting the microbiota of suppressive soils from an organic field site has succeeded in the identification of a clone with the ability to inhibit the growth of Bacillus subtilis DSM10. Sequencing of the fosmid identified a putative b-lactamase-like gene abgT. Transposon mutagenesis of the abgT gene resulted in a loss in ability to inhibit the growth of B. subtilis DSM10. Further analysis of the deduced amino acid sequence of AbgT revealed moderate homology to esterases, suggesting that the protein may possess hydrolytic activity. Weak lipolytic activity was detected; however the clone did not appear to produce any b-lactamase activity. Phylogenetic analysis revealed the protein is a member of the family VIII group of lipase/esterases and clusters with a number of proteins of metagenomic origin. The abgT gene was sub-cloned into a protein expression vector and when introduced into the abgT transposon mutant clones restored the ability of the clones to inhibit the growth of B. subtilis DSM10, clearly indicating that the abgT gene is involved in the antibacterial activity. While the precise role of this protein has yet to fully elucidated, it may be involved in the generation of free fatty acid with antibacterial properties. Thus functional metagenomic approaches continue to provide a significant resource for the discovery of novel functional proteins and it is clear that hydrolytic enzymes, such as AbgT, may be a potential source for the development of future antimicrobial therapies.

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Isolation and characterization of cold-active family VIII esterases from an arctic soil metagenome

Cited by 72 publications

References 27 publications

Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

Novel Metagenome-Derived Carboxylesterase That Hydrolyzes β-Lactam Antibiotics

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

Inhibition of the growth ofBacillus subtilisDSM10 by a newly discovered antibacterial protein from the soil metagenome

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