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Functional analysis of non-proteolytic enzymes involved in carbon-nitrogen bond cleavage

Research Project

Project/Area Number 14360047
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionUniversity of Tsukuba

Principal Investigator

KOBAYASHI Michihiko  University of Tsukuba, Graduate School of Life and Envirolmental Sciences, Professor, 大学院・生命環境科学研究科, 教授 (70221976)

Co-Investigator(Kenkyū-buntansha) HASHIMOTO Yoshiteru  University of Tsukuba, Graduate School of Life and Envirolmental Sciences, Assistant Professor, 大学院・生命環境科学研究科, 助手 (00323254)
HIGASHIBATA Hiroki  University of Tsukuba, Graduate School of Life and Envirolmental Sciences, Assistant Professor, 大学院・生命環境科学研究科, 助手 (20344864)
MASEDA Hideaki  University of Tsukuba, Graduate School of Life and Envirolmental Silences, Assistant Professor, 大学院・生命環境科学研究科, 助手 (10372343)
合田 昌彦  日本学術振興会, 応用生物化学系, 特別研究員(PD)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥14,700,000 (Direct Cost: ¥14,700,000)
Fiscal Year 2004: ¥4,400,000 (Direct Cost: ¥4,400,000)
Fiscal Year 2003: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2002: ¥6,100,000 (Direct Cost: ¥6,100,000)
KeywordsNitrilase / Nitrile hydratase / Amidase / Aldoxime dehydratase / Isonitrile hydtratase / N-substituted formamide deformylase / Nitrile / N-substituted formamide / アシルCoA合成酵素 / アルドキシムデヒドラターゼ / N-置換ホルムアシド分解酵素 / ニトリルイソニトリル / アミド / イソニトリルヒドラーゼ / アシド
Research Abstract

Among non-proteolytic enzymes involved in carbon-nitrogen bond cleavage, two enzymes involved in isonitrile metabolism are described as follows.
Isonitrile hydratase is a novel enzyme in Pseudomonas putida N19-2 that catalyzes the conversion of isonitriles to N-substituted formamides. Based on N-terminal and internal amino acid sequences, the isonitrile hydratase gene was cloned. The predicted amino acid sequence of inhA showed low similarity to that of an intracellular protease in Pyrococcus horikoshii (PH1704), and an active cysteine residue in the protease was conserved in the isonitrile hydratase at the corresponding position (Cys101). A mutant enzyme containing Ala instead of Cys101 did not exhibit isonitrile hydratase activity at all, demonstrating the essential role of this residue in the catalytic function.
N-Substituted formamide was produced through the hydration of an isonitrile by isonitrile hydratase in the isonitrile metabolism. The former compound was further degraded by a … More microorganism, F164, which was isolated from soil through an acclimatization culture. The N-substituted formamide-degrading microorganism was identified as Arthrobacter pascens. The microbial degradation was found to proceed through an enzymatic reaction, the N-substituted formamide being hydrolyzed to yield the corresponding amine and formate. The enzyme, designated as N-substituted formamide deformylase (NfdA), was purified and characterized. It stoichiometrically catalyzed the hydrolysis of N-benzylformamide (NBFA : an N-substituted formamide) to benzylamine and formate. Of all the N-substituted formamides tested, NBFA was the most suitable substrate for the enzyme. However, no amides were accepted as substrates. The gene (nfdA) encoding this enzyme was also cloned. The deduced amino acid sequence of nfdA exhibited the highest overall sequence identity (28%) with those of regulatory proteins among known proteins. Only the N-terminal region of NfdA also showed significant sequence identity (27-73%) to that of each member of the amidohydrolase superfamily, although there was no similarity in the overall sequence except in the above limited region. Less

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (32 results)

All 2005 2004 2003 2002 Other

All Journal Article (24 results) Publications (8 results)

  • [Journal Article] High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system2005

    • Author(s)
      Fukatsu, H., et al.
    • Journal Title

      Protein Expr.Purif. 40

      Pages: 212-219

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Optimum culture conditions for the production of N-substituted formamide deformylase by Arthrobacter pascens F1642005

    • Author(s)
      Fukatsu, H., et al.
    • Journal Title

      Biosci.Biotech.Biochem. 69

      Pages: 228-230

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Optimum culture conditions for the production of N-substituted formamide deformylase by Arthrobacter pascens F1642005

    • Author(s)
      Fukatsu, H., Goda, M., Hashimoto, Y., Higashibata, H., Kobayashi, M.
    • Journal Title

      Biosci.Biotech.Biochem. 69

      Pages: 228-230

    • NAID

      130000030579

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system2005

    • Author(s)
      Fukatsu, H., Herai, S., Hashimoto, Y., Maseda, H., Higashibata, H., Kobayashi, M.
    • Journal Title

      Protein Expr.Purif. 40

      Pages: 212-219

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Overall metabolic gene organization and characterization of the enzyme.2005

    • Author(s)
      Hashimoto, Y. et al.
    • Journal Title

      J.Biol.Chem. 280

      Pages: 8660-8667

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Stopped-flow Spectrophotometric and Resonance Raman Analyses of Aldoxine Dehydrafase Involved in Carbon-Nitrogen Triple Bond Synthesis.2005

    • Author(s)
      Oinuma, K-I. et al.
    • Journal Title

      FEBS Lett 579

      Pages: 1394-1398

    • Related Report
      2004 Annual Research Report
  • [Journal Article] High-level expression of a novel amine synthesizing enzyme, N-Subsistifuld formamide defomylase, in Streptomyces with a strong protein expression system2005

    • Author(s)
      Fukatsu, H. et al.
    • Journal Title

      Protein Expr.Purif. 40

      Pages: 212-219

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Optimum culture conditions for the production of N-Substituted formamide deformylase by Arthrobacter Pascens F1642005

    • Author(s)
      Fukatsu, H. et al.
    • Journal Title

      Biosci.Biotech.Biochem 69

      Pages: 228-230

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Amine-synthesizing enzyme N-substituted formamide deformylase : Screening, purification, characterization and gene cloning2004

    • Author(s)
      Fukatsu, H., et al.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 13726-13731

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase2004

    • Author(s)
      Konishi, K., et al.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 47619-47625

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Heme environment in aldoxime dehydratase involved in carbon- nitrogen triple bond synthesis2004

    • Author(s)
      Oinuma, K-I., et al.
    • Journal Title

      FEBS Lett. 568

      Pages: 44-48

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rhodococcus工業生産菌のニトリル分解酵素遺伝子群の解析と応用2004

    • Author(s)
      橋本義輝 ら
    • Journal Title

      日本農芸化学会誌 78

      Pages: 1073-1075

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase2004

    • Author(s)
      Konishi, K., Ishida, K., Oinuma, K-I., Ohta, T., Hashimoto, Y., Higashibata, H., Kitagawa, T., Kobayashi, M.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 47619-47625

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Amine-synthesizing enzyme N-substituted formamide deformylase : Screening, purification, characterization and gene cloning2004

    • Author(s)
      Fukatsu, H., Hashimoto, Y., Goda, M., Higashibata, H., Kobayashi, M.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 13726-13731

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Hyper-inducible expression system for streptomycetes2004

    • Author(s)
      Herai, S. et al.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 14031-14035

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Amine-synthesizing enzyme N-Substituted de formamide deformylase : Screening, purification, characterization and gene cloning2004

    • Author(s)
      Fukatsu, H. et al.
    • Journal Title

      Proc.Natl.Acad.Sci.USA 101

      Pages: 13726-13731

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification and characterization2003

    • Author(s)
      Oinuma, K-I., et al.
    • Journal Title

      J.Biol.Chem., 278

      Pages: 29600-29608

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] 新規酵素イソニトリルヒドラターゼの発見2003

    • Author(s)
      合田昌彦 ら
    • Journal Title

      バイオサイエンスとインダストリー 61

      Pages: 469-472

    • NAID

      10011624109

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification and characterization2003

    • Author(s)
      Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.
    • Journal Title

      J.Biol.Chem. 278

      Pages: 29600-29608

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] A novel lipase from Pseudomonas fluorescens HU380 : Gene cloning, overproduction, renaturation-activation, two-step purification and characterization2003

    • Author(s)
      Kojima, Y., Kobayashi, M., Shimizu, S.
    • Journal Title

      J.Biosci.Bioeng. 96

      Pages: 242-249

    • NAID

      110002665185

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase2002

    • Author(s)
      Hashimoto, Y., et al.
    • Journal Title

      J.Inorg.Biochem. 91

      Pages: 70-77

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Isonitrile hydratase from Pseudomonas putida N19-2 : Cloning, sequencing, gene expression, and identification of its active amino acid residue2002

    • Author(s)
      Goda, M., et al.
    • Journal Title

      J.Biol.Chem. 277

      Pages: 45860-45865

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Isonitrile hydratase from Pseudomonas putida N19-2 : Cloning, sequencing, gene expression, and identification of its active amino acid residue2002

    • Author(s)
      Goda, M., Hashimoto, Y., Takase M., Herai, S., Iwahara, Y., Higashibata, H., Kobayashi, M.
    • Journal Title

      J.Biol.Chem. 277

      Pages: 45860-45865

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase2002

    • Author(s)
      Hashimoto, Y., Sasaki, S., Herai.S., Oinuma, K,.Shimizu, S., Kobayashi, M.
    • Journal Title

      J.Inorg.Biochem. 91

      Pages: 70-77

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Oinuma, K-I. et al.: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of pseudomonas chlororaphis B23 : Sequencing, gene expression purification and characterization"J.Biol.Chem.. 278. 29600-29608 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Kojima, Y. et al.: "A novel lipase from pseudomonas fluorescens HU380 : Gene cloning overproduction, renaturation-activation, two-step purification and characterization"J.Biosci.Bioeng.. 96. 242-249 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Shiraki, K. et al.: "Genetic, enzymatic and structural analyses of phenylalanyl-tRNA synthetase from Thermococcus kodakaraensis koD1"J.Biochem.. 134. 567-574 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Higashibata, H. et al.: "Helicase and muclease activities of hyperthermophile pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5'-end"J.Biol.Chem.. 278. 15983-15990 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Higashibata H. et al.: "Surface histidine residue of archaeal histone affects DNA compaction and thermostability"FEMS.Microbiol.Lett.. 224. 17-22 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Goda, M.et al.: "Isonitrile hydrotase from Psundomonas putida N19-2"J.Biol.Chem.. 277. 45860-45865 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hashimoto, Y.et al.: "Site directed mntagenesis for cysteine residues of cobalt-containing nitrile hydratase"J.Inorg.Biochem.. 91. 70-77 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Higashibata, H.et al.: "Helicase and endonuclease activities of hyperthermophile Pynococcus horikoshii Dua2 inhibited by substrates with RNA segnlents at 5'-end"J.Biol.Chem.. (印刷中). (2003)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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