Reversible and nonoxidative γ-resorcylic acid decarboxylase: Characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

Yoshitaka Ishii, Yoshiki Narimatsu, Yuichiro Iwasaki, Naoki Arai, Kuniki Kino, Kotaro Kirimura

    Research output: Contribution to journalArticle

    37 Citations (Scopus)

    Abstract

    We found a γ-resorcylic acid (γ-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form γ-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of γ-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of γ-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with γ-RA. The enzyme, γ-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of γ-RA, but not α-RA or β-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form γ-RA, without formation of α-RA and β-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this γ-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of γ-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the γ-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of γ-RA and carboxylation of RE.

    Original languageEnglish
    Pages (from-to)611-620
    Number of pages10
    JournalBiochemical and Biophysical Research Communications
    Volume324
    Issue number2
    DOIs
    Publication statusPublished - 2004 Nov 12

    Fingerprint

    Carboxylation
    Agrobacterium tumefaciens
    Carboxy-Lyases
    Cloning
    Organism Cloning
    Decarboxylation
    Genes
    Acids
    Enzymes
    Hydrophobic chromatography
    Polyacrylamide Gel Electrophoresis
    Sphingomonas
    Amino Acids
    Mutagenesis
    Gene encoding
    Aspergillus niger
    Aspergillus
    Coenzymes
    Ion Exchange Chromatography
    Site-Directed Mutagenesis

    Keywords

    • γ-Resorcylic acid
    • 1,3-Dihydroxybenzene
    • 2,6-Dihydroxybenzoic acid
    • Agrobacterium tumefaciens
    • Nonoxidative decarboxylase
    • Regio-selective carboxylation
    • Resorcinol
    • Reversible decarboxylase
    • Rhizobium radiobacter

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Molecular Biology

    Cite this

    @article{811572b419f5491abc654845ef0b35cc,
    title = "Reversible and nonoxidative γ-resorcylic acid decarboxylase: Characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter",
    abstract = "We found a γ-resorcylic acid (γ-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form γ-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of γ-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of γ-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with γ-RA. The enzyme, γ-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of γ-RA, but not α-RA or β-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form γ-RA, without formation of α-RA and β-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this γ-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of γ-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the γ-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42{\%} and 30{\%} identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of γ-RA and carboxylation of RE.",
    keywords = "γ-Resorcylic acid, 1,3-Dihydroxybenzene, 2,6-Dihydroxybenzoic acid, Agrobacterium tumefaciens, Nonoxidative decarboxylase, Regio-selective carboxylation, Resorcinol, Reversible decarboxylase, Rhizobium radiobacter",
    author = "Yoshitaka Ishii and Yoshiki Narimatsu and Yuichiro Iwasaki and Naoki Arai and Kuniki Kino and Kotaro Kirimura",
    year = "2004",
    month = "11",
    day = "12",
    doi = "10.1016/j.bbrc.2004.09.091",
    language = "English",
    volume = "324",
    pages = "611--620",
    journal = "Biochemical and Biophysical Research Communications",
    issn = "0006-291X",
    publisher = "Academic Press Inc.",
    number = "2",

    }

    TY - JOUR

    T1 - Reversible and nonoxidative γ-resorcylic acid decarboxylase

    T2 - Characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

    AU - Ishii, Yoshitaka

    AU - Narimatsu, Yoshiki

    AU - Iwasaki, Yuichiro

    AU - Arai, Naoki

    AU - Kino, Kuniki

    AU - Kirimura, Kotaro

    PY - 2004/11/12

    Y1 - 2004/11/12

    N2 - We found a γ-resorcylic acid (γ-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form γ-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of γ-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of γ-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with γ-RA. The enzyme, γ-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of γ-RA, but not α-RA or β-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form γ-RA, without formation of α-RA and β-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this γ-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of γ-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the γ-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of γ-RA and carboxylation of RE.

    AB - We found a γ-resorcylic acid (γ-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form γ-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of γ-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of γ-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with γ-RA. The enzyme, γ-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of γ-RA, but not α-RA or β-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form γ-RA, without formation of α-RA and β-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this γ-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of γ-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the γ-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of γ-RA and carboxylation of RE.

    KW - γ-Resorcylic acid

    KW - 1,3-Dihydroxybenzene

    KW - 2,6-Dihydroxybenzoic acid

    KW - Agrobacterium tumefaciens

    KW - Nonoxidative decarboxylase

    KW - Regio-selective carboxylation

    KW - Resorcinol

    KW - Reversible decarboxylase

    KW - Rhizobium radiobacter

    UR - http://www.scopus.com/inward/record.url?scp=5144220809&partnerID=8YFLogxK

    UR - http://www.scopus.com/inward/citedby.url?scp=5144220809&partnerID=8YFLogxK

    U2 - 10.1016/j.bbrc.2004.09.091

    DO - 10.1016/j.bbrc.2004.09.091

    M3 - Article

    C2 - 15474471

    AN - SCOPUS:5144220809

    VL - 324

    SP - 611

    EP - 620

    JO - Biochemical and Biophysical Research Communications

    JF - Biochemical and Biophysical Research Communications

    SN - 0006-291X

    IS - 2

    ER -