Structural and functional analysis of anti-tumor microbial enzymes and their application for development of next generation anti-tutor enzymes.

• Project/Area Number: 18580076
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied microbiology
• Research Institution: Okayama University
• Principal Investigator: INAGAKI Kenji Okayama University, Graduate School of Natural Science and Technology, Prof (80184711)
• 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)
• Project Period (FY): 2006 – 2007
• Project Status: Completed (Fiscal Year 2007)
• Budget Amount: ¥3,890,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥390,000); Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
• Keywords: L-methionine γ-Lyase / L-lysine α-oxidase / structural & functional analysis / gene cloning / anti-tumor enzyme / L-メチオニンγ-リアーゼ / L-リジンα-オキシダーゼ / 抗腫瘍効果

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.

Research Products

• [Journal Article] Structure and quantum chemical analysis of NAD+ -dipendent isocitrate dehydrogenase: Hydride transfer and co-factor specificity (2008)
  • Author(s): Imada, K., Tamura, T., Takenaka, R., Kobayashi, I., Namba, K., Inagaki, K.
  • Journal Title: Proteins 70 Pages: 63-71
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] The Role of Cystein 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida (2008)
  • Author(s): Kudo, D., Misaki, S., Yamashita M., Tamura, T., Esaki, N., Inagaki, K.
  • Journal Title: Biosci. Biotech. Biochem. 72(印刷中)
  • NAID: 10027529566
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] Structure of the Antitumour Enzyme L-Methionine γ-Lyase from Pseudomonas putida at 1.8A Resolution (2008)
  • Author(s): Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takamura, T., Yoshioka, T., Yagi, S., Hoffman, M.R., Takimoto, A., Inagaki, K
  • Journal Title: J. Biochem 141 Pages: 535-54
  • NAID: 10020100176
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structure and quantum chemical analysis of NAD+ -dipendent isocitrate dehycirogenase : Hydride transfer and co-factor specificity (2008)
  • Author(s): Jmada, K., Tamura, T., Takenaka, R., Kobayashi, I., Namba, K., Inagaki, K
  • Journal Title: Proteins 70 Pages: 63-71
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] The Role of Cystein 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putida (2008)
  • Author(s): Kudo, D., Misaki, S., Yamashita M., Tamura. T., Esaki, N., Inagaki, K.
  • Journal Title: Biosci. Biotech. Biochem. 72(印刷中)
  • NAID: 10027529566
  • Peer Reviewed
• [Journal Article] Structure of the Antitumour Enzyme L-Methionine γ-Lyase from Pseudomonas putida at 1.8A Resolution (2007)
  • Author(s): Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takamura, T., Yoshioka, T., Yagi, S., Hoffman, M. R., Takimoto, A., Inagaki, K.
  • Journal Title: J. Biochem. 141 Pages: 535-544
  • NAID: 10020100176
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] 抗腫瘍性酵素L-メチオニン γ-リアーゼの構造機能解析 (2007)
  • Author(s): 工藤大蔵, 稲垣賢二
  • Journal Title: 生化学 79 Pages: 682-686
  • NAID: 10019730174
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] 植物由来のL-メチオニン γ-リアーゼの性質と機能 (2007)
  • Author(s): 工藤大蔵, 稲垣賢二
  • Journal Title: ビタミン 81 Pages: 497-499
  • NAID: 110006449220
  • Description: 「研究成果報告書概要(和文)」より
  • Peer Reviewed
• [Journal Article] The Role of Cystein 116 in the Active Site of the Antitumor Enzyme L-Methionine γ-Lyase from Pseudomonas putjda (2007)
  • Author(s): Kudo, D., Misaki, S., Yamashita, M., Tamura, T., Esaki, N., Inagaki, K
  • Journal Title: Biosci. Biotech. Biochem 72(in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structural and functional analysis of Antitumor Enzyme L-Methionine γ-Lyase (2007)
  • Author(s): Kudo, D., Inagaki, K
  • Journal Title: SEIKAGAKU 79 Pages: 682-686
  • NAID: 10019730174
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Characteristics and function of L-Methionine γ-Lyase from plants (2007)
  • Author(s): Kudo, D., Inagaki, K
  • Journal Title: Vitamines 81 Pages: 497-499
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Tumor targeting proteins (2007)
  • Author(s): Inagaki, K., Tamura, T
  • Journal Title: Structural Biology(S. Kuramitsu & M. Sugiyama Eds.)(Kyoritsu Pub. Tokyo)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structure of the Antitumour Enzyme L-Methionine γ-Lyase from Pseudomonas putida at 1.8Å Resolution (2007)
  • Author(s): Kudou, D., Misaki, S., Yamashita, M., Tamura, T., Takamura, T., Yoshioka, T., Yagi, S., Hoffman, M. R., Takimoto, A., Inagaki, K.
  • Journal Title: J. Biochem. 141 Pages: 535-544
  • Peer Reviewed
• [Journal Article] High-level expression and bulk crystallization of recombinant L-methionine γ-lyase, an anticancer agent. (2006)
  • Author(s): Takakura, T, Ito, T, Yagi, S, Notsu, Y, Itakura, T, Nakamura, T, Inagaki, K, Esaki N, Hoffman, R.M, Takimoto,A
  • Journal Title: Appl.Microbiol.Biotechnol 70 Pages: 183-192
• [Journal Article] Physicochemical and Pharmacokinetic Charactcrization of Highly Potent Recombinant L-Methionine γ-Lyase Conjugated with Polyethylene Glycol as an Antitumor Agent. (2006)
  • Author(s): Takakura, T, Takimoto, A, Notsu, Y, Yoshida, H, Ito, T, Nagatome, H, Ohno, M, Kobayashi, Y, Yoshioka, T, Inagaki, K, Yagi, S, Hoffman, M.R, Esaki. N
  • Journal Title: Cancer Res 66 Pages: 2807-2814
• [Journal Article] Characterization of Monofunctional Catalase KatA from Radioresistant Bacterium Deinococcus radiodurans. (2006)
  • Author(s): Kobayashi, I, Tamura, T, Sghaicr, H, Narumi, I, Yamaguchi, S, Umeda, K, Inagaki,K
  • Journal Title: J.Biosci.Bioeng 101 Pages: 315-321
  • NAID: 110004736889
• [Journal Article] Effects of the Principal Lovastatin Production by Monascus Pilosus : Nutrients on. (2006)
  • Author(s): Miyake, T, Uchitomi, K, Zhang, M.Y, Kono, I, Nozaki, N, Sammoto, H, Inagaki,K
  • Journal Title: Biosci.Biotechnol.Biochem 70 Pages: 1154-1159
• [Journal Article] The Crystal Structure of Hypothetical Methyltransferase from Thermus Thermophilus HB8. (2006)
  • Author(s): Sasaki, C, Sugiura, I, Ebihara, A, Tamura, T, Sugio, S, Inagaki,K
  • Journal Title: PROTEINS : Structure,Function,and Bioinformatics 64 Pages: 552-557
• [Journal Article] Performance characterization of recombinant L-glutamate oxidese in a micro GOT/GPT sensing system. (2006)
  • Author(s): Upadhyay, S, Ohgami, N, Kusakabe, H, Mizuno, H, Arima, J, Tamura, T, Inagaki, I, Suzuki,H
  • Journal Title: Sensor and Actuators B 119 Pages: 570-576
• [Presentation] 抗腫瘍性酵素L-リジン α-オキシダーゼの遺伝子クローニング (2007)
  • Author(s): 中田春香, 田村隆, 稲垣純子, 日下部均, 稲垣賢二
  • Organizer: 日本ビタミン学会第59回大会
  • Place of Presentation: 長崎
  • Year and Date: 2007-05-25
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Gene cloning of anti-tumor enzyme L-lysine α-oxidase (2007)
  • 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
• [Presentation] 抗腫瘍性酵素L-リジンα-オキシダーゼの遺伝子クローニング (2007)
  • Author(s): 中田春香, 田村隆, 稲垣純子, 稲垣賢
  • Organizer: 日本ビタミン学会第59回大会
  • Place of Presentation: 長崎
  • Year and Date: 2007-05-25
• [Presentation] L-Methionine γ-Lyaseの活性中心残基Cys116への変異導入による基質特異性の改変 (2007)
  • Author(s): 工藤大蔵, 田村隆, 岬真太郎, 瀧本明生, 稲垣賢二
  • Organizer: 日本農芸化学会中四国支部第18回講演会
  • Place of Presentation: 広島
  • Year and Date: 2007-05-12
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] Change of substrate supecificity of residues in the active site of L-Methionine γ-Lyase (2007)
  • 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
• [Presentation] L-Methionine γ-Lyaseの活性中心残基Cys116への変異導入による基質特異性の改変 (2007)
  • Author(s): 工藤大蔵, 田村隆, 岬真太郎, 稲垣賢二
  • Organizer: 日本農芸化学会 中四国支部第18回講演会
  • Place of Presentation: 広島
  • Year and Date: 2007-05-12
• [Presentation] 抗腫瘍性酵素L-メチオニン γ-リアーゼ活性中心に存在するCys残基の機能解析 (2007)
  • Author(s): 工藤大蔵, 田村隆, 岬真太郎, 稲垣賢二
  • Organizer: 日本農芸化学会2007年度大会
  • Place of Presentation: 東京
  • Year and Date: 2007-03-25
  • Description: 「研究成果報告書概要(和文)」より
• [Presentation] The functional analysis of Cys residues in active site of L-Methionine γ-Lyase (2007)
  • Author(s): Kudo, D., Tamura, T., Misaki, S., Inagaki, K
  • Organizer: Japan Society for Bioscience. Biotechnology, and Agrochemistry. annual meeting 2007
  • Place of Presentation: Tokyo
  • Year and Date: 2007-03-25
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] 構造生物学-ポストゲノム時代のタンパク質- (2007)
  • Author(s): 稲垣 賢二, 他(共著)
  • Total Pages: 259
  • Publisher: 共立出版
  • Description: 「研究成果報告書概要(和文)」より
• [Book] 構造生物学-ポストゲノム時代のタンパク質研究- (2007)
  • Author(s): 稲垣 賢二, 他(共著)
  • Total Pages: 261
  • Publisher: 共立出版