2007 Fiscal Year Final Research Report Summary
Structural and functional analysis of anti-tumor microbial enzymes and their application for development of next generation anti-tutor enzymes.
| Project/Area Number |
18580076
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Okayama University |
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Principal Investigator |
INAGAKI Kenji Okayama University, Graduate School of Natural Science and Technology, Prof (80184711)
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| Co-Investigator(Kenkyū-buntansha) |
TAMURA Takashi Okayama University, Graduate School of Natural Science and Technology, associate prof (40253009)
INAGAKI Junko Okayama University, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, assistant prof (90271056)
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| Project Period (FY) |
2006 – 2007
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| Keywords | L-methionine γ-Lyase / L-lysine α-oxidase / structural & functional analysis / gene cloning / anti-tumor enzyme |
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| Research Abstract |
A highly potent recombinant L-methionine γ-lyase from Pseudomonas putida (MGL_Pp. EC 4.4.1.11) has been characterized physicochemically and pharmacokinetically in vivo and in vitro as a potent anticancer agent that can deplete L-methionine from plasma. The detailed structure of MGL_Pp and the function of the active site residue have so far not been cleared to improve as a next generation antitumor enzyme. We demonstrated that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method at 1.8A resolution. Detailed information of the overall structure of MGL_Pp supplied clear pictures of the N-terminal domain and the substrate-and PLP-binding pockets. It was found that a hydrogen bond network was formed around a cofactor pyridoxal 5'-phosphate in the MGL active site, which is specific for MGLs. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug. The 3D structure of MGL_Pp suggested that Cys116 might be involved in the hydrogen bond network at the active site. We found that Cys116 plays an important role in the γ-elimination reaction of L-methionine and for the substrate recognition in the MGLs. We also discovered that the substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a change of the substrate specificity, suggesting that the His residue may be directly involved in the γ-elimination reaction. With a low purity, it was found that MGL_Pp was degraded between Cys49-Phe50 and the degraded enzyme lost the activity. Conjugation of MGL_Pp with polyethylene glycol (PEG) will importantly reduce proteolysis. Recently, the complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined. The gene encoding L-methionine γ-lyase from Streptomyces avermitilis was cloned and expressed in Escherichia coli to characterize the enzymological properties of the gene product. MGL_Sa. for the first time over the world.
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Research Products
(18 results)
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[Journal Article] Structure and quantum chemical analysis of NAD+ -dipendent isocitrate dehydrogenase: Hydride transfer and co-factor specificity2008
Author(s)
Imada, K., Tamura, T., Takenaka, R., Kobayashi, I., Namba, K., Inagaki, K.
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Journal Title
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] The Role of Cystein 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida2008
Author(s)
Kudo, D., Misaki, S., Yamashita M., Tamura, T., Esaki, N., Inagaki, K.
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Journal Title
Biosci. Biotech. Biochem. 72(印刷中)
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Structure of the Antitumour Enzyme L-Methionine γ-Lyase from Pseudomonas putida at 1.8A Resolution2008
Author(s)
Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takamura, T., Yoshioka, T., Yagi, S., Hoffman, M.R., Takimoto, A., Inagaki, K
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Journal Title
J. Biochem 141
Pages: 535-54
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Structure and quantum chemical analysis of NAD+ -dipendent isocitrate dehycirogenase : Hydride transfer and co-factor specificity2008
Author(s)
Jmada, K., Tamura, T., Takenaka, R., Kobayashi, I., Namba, K., Inagaki, K
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Journal Title
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Structure of the Antitumour Enzyme L-Methionine γ-Lyase from Pseudomonas putida at 1.8A Resolution2007
Author(s)
Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takamura, T., Yoshioka, T., Yagi, S., Hoffman, M. R., Takimoto, A., Inagaki, K.
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Journal Title
J. Biochem. 141
Pages: 535-544
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] The Role of Cystein 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putjda2007
Author(s)
Kudo, D., Misaki, S., Yamashita, M., Tamura, T., Esaki, N., Inagaki, K
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Journal Title
Biosci. Biotech. Biochem 72(in press)
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Gene cloning of anti-tumor enzyme L-lysine α-oxidase2007
Author(s)
Nakata, H., Tamura, T., Inagaki, J., Kusakabe, H., Inagaki, K
Organizer
The Vitamin Society of Japan, 59th annual meeting
Place of Presentation
Nagasaki
Year and Date
2007-05-25
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Change of substrate supecificity of residues in the active site of L-Methionine γ-Lyase2007
Author(s)
Kudo, D., Tamura, T., Misaki, S., Takimoto, A., Inagaki, K
Organizer
Tyu-shikoku Branch Seminar No.18. Japan Society for Bioscience, Biotechnology, and Agrochemistry
Place of Presentation
Hiroshima
Year and Date
2007-05-12
Description
「研究成果報告書概要(欧文)」より
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