活性酸素種による遺伝子発現誘導の分子基盤

• Project/Area Number: 12147208
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Nippon Medical School
• Principal Investigator: 西野 武士 日本医科大学, 大学院・医学研究科, 教授 (40094312)
• Co-Investigator(Kenkyū-buntansha): 阿部 靖子 日本医科大学, 医学部, 講師 (60089612) ; 松村 智裕 日本医科大学, 医学部, 助手 (20297930) ; 片山 映 日本医科大学, 医学部, 助手 (10333113)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥55,400,000 (Direct Cost: ¥55,400,000); Fiscal Year 2004: ¥13,800,000 (Direct Cost: ¥13,800,000); Fiscal Year 2003: ¥13,600,000 (Direct Cost: ¥13,600,000); Fiscal Year 2002: ¥14,000,000 (Direct Cost: ¥14,000,000); Fiscal Year 2001: ¥14,000,000 (Direct Cost: ¥14,000,000)
• Keywords: 活性酸素 / キサンチン脱水素酵素 / キサンチン酸化酵素 / 金属タンパク質 / X線結晶解析 / 非ヘム鉄 / ペルオキレドキシン / 鉄結合タンパク質 / ペルオキシレドキシン

Research Abstract

1)スーパーオキシドおよび過酸化水素それぞれの生成酵素の発現系の開発と誘導機構とスーパーオキシドセンサーの探索:前年に引き続きキサンチン脱水素酵素のモリブデン近傍の活性中心に阻害剤であるFYX-051の立体構造をX線結晶解析を進めた。また結合にともない長波吸収体を観察し、モリブデン配位子のジオメトリー決定をおこなった。これらの情報を基に基本的なモリブデン水酸化機構の反応機構の全貌を明らかにした。さらに脱水素酵素から酸化酵素への変換に関与するトリガーの最終同定と変換しないラット酵素(活性酸素非産生型)のX-線結晶構造解析、また殆どスーパーオキシドのみを産生する変異体の二つの型のX-線結晶構造を解析した。これらの情報からXDHからXO型酵素への変換機構をほぼ全貌を解明させた。これら(水酸化機構の解明、および活性酸素生成酵素への変換の分子機構の解明)は本酵素に関する永年の歴史的な課題であるが、その解明は本特定研究による大きな成果である。さらに生理的意義を解明すべき、変換させる因子の牛乳からの抽出同定をおこなった。また活性酸素の細胞化学的意義の解明をめざし細胞内での上記2つの変異酵素の発現を試みた。
2)金属結合蛋白の同定:前年に引き続き、鉄結合タンパク質の検索をめざして、培養細胞を用いてポリアクリルアミドゲル二次元電気泳動で分画し、鉄結合タンパク質のデータバンク作成にむけて、既知鉄タンパク質のスポット同定を引き続き進めた。また人胎盤蛋白質の解析を試みた。
3)箱嶋らと共同でおこなったHBP23の立体構造の解析結果をもとに、活性中心のCys52、Cys173およびその周辺のアミノ酸残基の変異体の解析、還元体であるチオレドキシンとの相互作用を引き続き解析し、さらにオリゴマー形成の酵素反応への影響を解析した。

Research Products

• [Journal Article] Inter-molecular electron transfer mechanism of the extracellular flavocytochrome cellobiose dehydrogenase from the basidiomycete Phanerochaete chrysosporium (2005)
  • Author(s): Kiyohiko Igarashi et al.
  • Journal Title: Eur.J.Biochem. (in press)
• [Journal Article] The crystal structure of xanthine oxidoreductase during catalysis : Implications for reaction mechanism and enzyme inhibition (2004)
  • Author(s): Ken Okamoto et al.
  • Journal Title: Proc.Natl.Acad.Sci.USA. 101・21 Pages: 7931-7936
• [Journal Article] Characterization and Molecular Cloning of Cellobiose Dehydrogenase from the Brown-Rot Fungus Coniophora puteana (2004)
  • Author(s): Taira Kajisa et al.
  • Journal Title: Journal of Bioscience and Bioengineering 98・1 Pages: 57-63
• [Journal Article] Y-700 [1-[3-Cyano-4-(2,2dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic Acid] : A Potent Xanthine Oxidoreductase Inhibitor with Hepatic Excretion (2004)
  • Author(s): Atsushi Fukunari et al.
  • Journal Title: The Journal of Pharmacorogy and Experimental Therapeutics 311 Pages: 519-528
• [Journal Article] The Role of Active Site Glutamate Residues in Catalysis of Rhodobacter capsulatus Xanthine Dehydrogenase (2004)
  • Author(s): Silke Leimkuhler et al.
  • Journal Title: J.Biol.Chem. 279・39 Pages: 40437-40444
• [Journal Article] キサンチン脱水素酵素から酸化酵素への蛋白質構造・機能の変換の分子機構 (2004)
  • Author(s): 岡本 研 他
  • Journal Title: 蛋白質 核酸 酵素 49・5 Pages: 625-633
• [Publications] Kuwabara, Y. et al.: "Unique relay system for switching from dehydrogenase to oxidase form of xanthine oxidoreductase"Proc.Natl.Acad.Sci.USA.. 100. 8170-8175 (2003)
• [Publications] Okamoto, K. et al.: "An Extremely Potent Inhibitor of Xanthine Oxidoreductase : Crystal Structure of the Enzyme-Inhibitor Complex and Mechanism of Inhibition"J.Biol.Chem.. 278. 1848-1855 (2003)
• [Publications] Maiti.et al.: "Resonance Raman Studies on Xanthine oxidase : observation of the Mo(IV)-ligand vibration."J.Biolo.Inorganic Chemistry. 8. 327-333 (2003)
• [Publications] 岡本研, 桑原允光, 西野朋子, 西野武士: "キサンチン脱水素酵素から酸化酵素への蛋白質構造・機能変換の分子機構"蛋白質・核酸・酵素. 49. 625-633 (2004)
• [Publications] 川本進, 西野朋子, 西野武士: "「ゲノミクス・プロテオミクスの新展開〜生物情報の解析と応用〜」バキュロウイルス/昆虫細胞によるタンパク質の発現と精製"エヌ・テイ・エス(東京). (2004)
• [Publications] K.Igarashi I.et al.(Nishino, T): "Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium"Biochem. J. 365. 521-526 (2002)
• [Publications] K.Akira, T.Atsuko, A.Wada, T.Nishino, A.Ishihama.: "Systematic search for zinc-binding proteins in Esherichia coli"Eur. J. Biochem.. 269. 2403-2413 (2002)
• [Publications] T.Nishino, Y.Amaya, S.Kawamoto, Y.Kashima, K.Okamato, T.Nishino: "Purification and Characterization of Multiple-Forms of Rat Liver Xanthine Oxidoreductase Expressed in Baculovirus-Insect Cell System"J. Biochem.. 132. 597-606 (2002)
• [Publications] Maiti NC, Tomita T, Kitagawa T, Okamoto K, Nishino T.: "Resonance Raman studies on xanthine oxidase : observation of Mo(VI)-ligand vibrations"J Biol Inorg Chem.. Feb;8(3). 327-333 (2003)
• [Publications] K.Okamoto, B.T.Eger, T.Nishino, S.Kondo, E.F.Pai, T.Nishino: "An Extremely Potent Inhibitor of Xanthine Oxidoreductase : Crystal Structure of the_Enzyme-Inhibitor Complex and Mechanism of Inhibition"J. Biol. Chem.. 278. 1848-1855 (2003)
• [Publications] E.Pai, T, Nishino: "The molybdenum-containing xanthine and picolic acid dehydrogenase"Metal Ions in Biological Systems (Text book, Eds.A.Siegel and H.Sigel),(Marcel Dekker, Inc., New York). 24 (2002)
• [Publications] Kusano T., Nishino To., Okamoto K., Hori H., Nishino T.: "The factor in bovine milk and the sulfhydryl residues of xanthine oxidoreductase responsible for conversion from dehydrogenase to oxidase forms"Flavins and Flavoproteins [Rudolf Weber Agency for Scentific Publications]. 4 (2002)
• [Publications] K.Ichida, T.Matsumura, R.Sakuma, T.Hosoya, T.Nishino: "Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II"Biochem. Biophys. Res. Commun.. 282. 1194-1200 (2001)
• [Publications] Cosper NJ, Scott RA, Hori H, Nishino T, Iwasaki T.: "X-ray absorption spectroscopic analysis of the high-spin ferriheme site in substrate-bound neuronal nitric-oxide synthase"J Biochem. 130(2). 191-198 (2001)
• [Publications] Nishino, T.: "Crystal structures of bovine milk xanthine dehydrogenase and mechanism of conversion from the dehydrogenase to the oxidase"Indian J. Chem. 41. 78-79 (2002)
• [Publications] Nishino, T., K.Okamoto, K., Pai, E.: "High-Resolution Structure of Bovine Milk Xanthine Oxidoreductase and Inhibitor Complexes"SPring-8 Research Frontiers. (in press).
• [Publications] Igarashi, I., Momohara, I., Nishino, T., Samejima, M.: "Kinetics of inter-domain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium"Biochem. J. (in press).
• [Publications] Pai, E., Nishino, T.: "Metal Ions in Biological Systems"Marcel Dekker, Inc., New York. 24 (2002)
• [Publications] T.Iwasaki,K.Okamoto,T.Nishino,J.Mizushima,H.Hori,and T.Nishino.: "Sequence Motif-specific Assignment of Two [2Fe-2S] Clusters in Rat Xanthine Oxidoreductase Studied by Site-directed Mutagenesis"J.Biochem.. 127. 771-778 (2000)
• [Publications] T.Kitagawa,M.Morisima,T.Nishino,and E.Kimura.: "(Review) Progress on "Molecular Biometallics" as a Project of the Priority Areas for Research under the Auspices of Japanese Government"J.Biol.Inorg.Chem.. 5. 410-415 (2000)
• [Publications] T.Nishino and K.Okamoto.: "(Reveiew) The Role of the [2Fe-2S] Cluster Centers in Xanthine Oxidoreductase."J.Inorg.Biochem.. 82. 43-49 (2000)
• [Publications] C.Enroth,B.T.Eger,K.Okamoto,T.Nishino,T.Nishino,and E.F.Pai.: "Crystal Structures of Bovine Milk Xanthine Dehydrogenase and Xanthine Oxidase : Structure-Based Mechanism of Conversion"Proc.Natle.Acad.Sci.,USA. 97. 10723-10728 (2000)
• [Publications] B.T.Eger,K.Okamoto,C.Enroth,M.Sato,T.Nishino,E.F.Pai,and T.Nishino.: "Purification, crystallization and preliminary X-ray diffraction studies of xanthine Dehydrogenase and xanthine Oxidase isolated from bovine milk."Acta Cryst.. D56. 1656-1658 (2000)
• [Publications] J.Park,T.Higuchi,K.Kikuchi1,Y.Urano,H.Hori,T.Nishino,J.Aoki,K.Inoue & T.Nagano: "Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides."Brithish J.Pharm.. 274(in press).
• [Publications] 広津晶子,阿部靖子,西野武士,箱嶋敏雄: "ペルオキシレドキシンの分子機能の構造的基盤"蛋白質・核酸・酵素. 45. 2463-2474 (2000)