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Characterization of a Novel Esterase Est33 From an Antarctic Bacterium: A Representative of a New Esterase Family.

ABSTRACT: Studies of microorganisms from extreme environments can sometimes reveal novel proteins with unique properties. Here, we identified a novel esterase gene (Est33) from an Antarctic bacterium. The protein was expressed and purified for biochemical characterizations. Site-mutation variants including S94A, D205A, and H233A were constructed to explore the structure-function relationship of the catalytic triad of Est33, and we found mutating Ser⁹⁴, Asp²⁰⁵, and His²³³ residues lead to a complete loss of enzyme activity. In addition, the catalytic Ser⁹⁴ located in a conserved pentapeptide motif GVSWG. Phylogenetic analysis showed that Est33 and its closely related homologs belonged to an independent group apart from other known family members, indicating that Est33 represented a new family of esterase. The Est33 enzyme was found to be a cold-active esterase retaining 25%-100% activity from 10°C to 30°C and to have optimal catalytic activity toward p-nitrophenol acetate (30°C and pH7.5). The serine modifying reagent phenylmethylsulfonyl fluoride inhibited the activity of Est33 by 77.34%, while thiol reagents such as dithiol threitol (DTT) activated the enzyme by 3-fold. Metal chelating reagents EDTA had no effects, indicating that Est33 is not a metalloenzyme. Collectively, these results indicate that Est33 constitutes the first member of a novel esterase family XXI that has been identified.

SUBMITTER: Liu X

PROVIDER: S-EPMC9152352 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Characterization of a Novel Esterase Est33 From an Antarctic Bacterium: A Representative of a New Esterase Family.

Liu Xiaoyu X Zhou Mingyang M Sun Rui R Xing Shu S Wu Tao T He Hailun H Chen Jianbin J Bielicki John Kevin JK

Frontiers in microbiology 20220517

Studies of microorganisms from extreme environments can sometimes reveal novel proteins with unique properties. Here, we identified a novel esterase gene (Est33) from an Antarctic bacterium. The protein was expressed and purified for biochemical characterizations. Site-mutation variants including S94A, D205A, and H233A were constructed to explore the structure-function relationship of the catalytic triad of Est33, and we found mutating Ser94, Asp205, and His233< ...[more]

PMID: 35655995

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Project description:BackgroundMicrobes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries.ResultsFunctional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di- and monoesters of alkyl-, aryl- and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases.ConclusionsThis study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalate-based plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Dataset Information

Characterization of a Novel Esterase Est33 From an Antarctic Bacterium: A Representative of a New Esterase Family.

Publications

Characterization of a Novel Esterase Est33 From an Antarctic Bacterium: A Representative of a New Esterase Family.

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