2004 Fiscal Year Final Research Report Summary
Functional analysis of non-proteolytic enzymes involved in carbon-nitrogen bond cleavage
| Project/Area Number |
14360047
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | University of Tsukuba |
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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)
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| 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)
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| Project Period (FY) |
2002 – 2004
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| Keywords | Nitrilase / Nitrile hydratase / Amidase / Aldoxime dehydratase / Isonitrile hydtratase / N-substituted formamide deformylase / Nitrile / N-substituted formamide |
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| 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
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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
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[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.
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Journal Title
Protein Expr.Purif. 40
Pages: 212-219
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
J.Biol.Chem. 279
Pages: 47619-47625
Description
「研究成果報告書概要(欧文)」より
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[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.
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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
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[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.
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Journal Title
J.Biol.Chem. 278
Pages: 29600-29608
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
J.Biol.Chem. 277
Pages: 45860-45865
Description
「研究成果報告書概要(欧文)」より
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[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.
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Journal Title
J.Inorg.Biochem. 91
Pages: 70-77
Description
「研究成果報告書概要(欧文)」より
