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Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

ABSTRACT: The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1?6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension (LNTE, residues 26-283) between a putative signal peptide (residues 1-25) and a C-terminal esterase domain (residues 284-510). A putative esterase from Candidatus Solibacter usitatus (CSu-Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC-Est1. To examine whether LC-Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC-Est1 (residues 26-510), LC-Est1C (residues 284-510), and LC-Est1C* (residues 304-510) were overproduced in E. coli, purified, and characterized. LC-Est1C* was only used for structural analysis. The crystal structure of LC-Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC-Est1C was lower than that of LC-Est1 by 60%, although its substrate specificity was similar to that of LC-Est1. LC-Est1C was less stable than LC-Est1 by 3.3°C. These results suggest that LNTE of LC-Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.

SUBMITTER: Okano H

PROVIDER: S-EPMC4282415 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

Okano Hiroyuki H Hong Xun X Kanaya Eiko E Angkawidjaja Clement C Kanaya Shigenori S

Protein science : a publication of the Protein Society 20141205 1

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1∼6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension ...[more]

PMID: 25348365

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Project description:BackgroundPyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes) and developing various biotechnological applications.ResultsThe pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45-49%). The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3), purified and characterized. The molecular mass of the native enzyme was approximately 31 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of the Pye3 indicated molecular mass of 31 kDa and 31.5 kDa, respectively, suggesting that the Pye3 is a monomer. The purified Pye3 not only degraded all pyrethroid pesticides tested, but also hydrolyzed rho-nitrophenyl esters of medium-short chain fatty acids, indicating that the Pye3 is an esterase with broader specificity. The Km values for trans-Permethrin and cis-permethrin are 0.10 muM and 0.18 muM, respectively, and these catalytic properties were superior to carboxylesterases from resistant insects and mammals. The catalytic activity of the Pye3 was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, whereas less pronounced effect was observed in the presence of divalent cations, the chelating agent EDTA and phenanthroline.ConclusionA novel pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, the broader substrate specificities and higher activity of the pyrethroid-hydrolyzing esterase (Pye3) make it an ideal candidate for in situ for detoxification of pyrethroids where they cause environmental contamination problems. Consequently, metagenomic DNA clone library offers possibilities to discover novel bio-molecules through the expression of genes from uncultivated bacteria.

Dataset Information

Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

Publications

Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

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