Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701

Kahori Yuhara, Hiromi Yonehara, Takasumi Hattori, Keiichi Kobayashi, Kotaro Kirimura

    Research output: Contribution to journalArticle

    4 Citations (Scopus)

    Abstract

    trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. Database The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980 It remains unclear how trans-aconitic acid is degraded and/or assimilated in vivo by plants. Here, Yuhara and colleagues report the identification and characterization of aconitate D-isomerase (AI, EC 5.3.3.7), and its encoding gene (ais). This new bacterial enzyme, which catalyses the isomerization between cis- and trans-aconitic acid, was purified to homogeneity from Pseudomonas sp. WU-0701 - a soil bacterium that assimilates trans-aconitic acid as a sole carbon source. Interestingly, the amino acid sequence of AI shows high homology with those of molybdenum ABC transporter substrate-binding proteins. Thus, this work has important implications for studies of tricarboxylic acid metabolism in nature.

    Original languageEnglish
    Pages (from-to)4257-4267
    Number of pages11
    JournalFEBS Journal
    Volume282
    Issue number22
    DOIs
    Publication statusPublished - 2015 Nov 1

    Fingerprint

    Aconitic Acid
    Isomerases
    Pseudomonas
    Genes
    Enzymes
    aconitate delta-isomerase
    Nucleic Acid Databases
    Amino Acids
    Amino Acid Sequence
    NSC 153174
    Gene encoding
    ATP-Binding Cassette Transporters
    Molybdenum
    Cell Extracts
    Purification
    Carrier Proteins
    Soil
    Carbon
    Nucleotides
    Tricarboxylic Acids

    Keywords

    • aconitate isomerase
    • citric acid
    • molybdenum ABC transporter
    • Pseudomonas sp.
    • trans-aconitic acid

    ASJC Scopus subject areas

    • Biochemistry
    • Cell Biology
    • Molecular Biology

    Cite this

    Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701. / Yuhara, Kahori; Yonehara, Hiromi; Hattori, Takasumi; Kobayashi, Keiichi; Kirimura, Kotaro.

    In: FEBS Journal, Vol. 282, No. 22, 01.11.2015, p. 4257-4267.

    Research output: Contribution to journalArticle

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    abstract = "trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90{\%} and 74{\%} identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. Database The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980 It remains unclear how trans-aconitic acid is degraded and/or assimilated in vivo by plants. Here, Yuhara and colleagues report the identification and characterization of aconitate D-isomerase (AI, EC 5.3.3.7), and its encoding gene (ais). This new bacterial enzyme, which catalyses the isomerization between cis- and trans-aconitic acid, was purified to homogeneity from Pseudomonas sp. WU-0701 - a soil bacterium that assimilates trans-aconitic acid as a sole carbon source. Interestingly, the amino acid sequence of AI shows high homology with those of molybdenum ABC transporter substrate-binding proteins. Thus, this work has important implications for studies of tricarboxylic acid metabolism in nature.",
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    T1 - Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701

    AU - Yuhara, Kahori

    AU - Yonehara, Hiromi

    AU - Hattori, Takasumi

    AU - Kobayashi, Keiichi

    AU - Kirimura, Kotaro

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    N2 - trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. Database The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980 It remains unclear how trans-aconitic acid is degraded and/or assimilated in vivo by plants. Here, Yuhara and colleagues report the identification and characterization of aconitate D-isomerase (AI, EC 5.3.3.7), and its encoding gene (ais). This new bacterial enzyme, which catalyses the isomerization between cis- and trans-aconitic acid, was purified to homogeneity from Pseudomonas sp. WU-0701 - a soil bacterium that assimilates trans-aconitic acid as a sole carbon source. Interestingly, the amino acid sequence of AI shows high homology with those of molybdenum ABC transporter substrate-binding proteins. Thus, this work has important implications for studies of tricarboxylic acid metabolism in nature.

    AB - trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. Database The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980 It remains unclear how trans-aconitic acid is degraded and/or assimilated in vivo by plants. Here, Yuhara and colleagues report the identification and characterization of aconitate D-isomerase (AI, EC 5.3.3.7), and its encoding gene (ais). This new bacterial enzyme, which catalyses the isomerization between cis- and trans-aconitic acid, was purified to homogeneity from Pseudomonas sp. WU-0701 - a soil bacterium that assimilates trans-aconitic acid as a sole carbon source. Interestingly, the amino acid sequence of AI shows high homology with those of molybdenum ABC transporter substrate-binding proteins. Thus, this work has important implications for studies of tricarboxylic acid metabolism in nature.

    KW - aconitate isomerase

    KW - citric acid

    KW - molybdenum ABC transporter

    KW - Pseudomonas sp.

    KW - trans-aconitic acid

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