発光細菌の発光反応関連酵素群のX線結晶構造解析

• Project/Area Number: 11169206
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The University of Tokyo
• Principal Investigator: 田之倉 優 東京大学, 大学院・農学生命科学研究科, 教授 (60136786)
• Co-Investigator(Kenkyū-buntansha): 佐々木 宏 常磐短期大学, 講師 (80272467) ; 永田 宏次 東京大学, 生物生産工学研究センター, 助手 (30280788)
• Project Period (FY): 1999 – 2002
• Project Status: Completed (Fiscal Year 2002)
• Budget Amount: ¥86,200,000 (Direct Cost: ¥86,200,000); Fiscal Year 2002: ¥12,200,000 (Direct Cost: ¥12,200,000); Fiscal Year 2001: ¥12,600,000 (Direct Cost: ¥12,600,000); Fiscal Year 2000: ¥21,400,000 (Direct Cost: ¥21,400,000); Fiscal Year 1999: ¥40,000,000 (Direct Cost: ¥40,000,000)
• Keywords: 生物発光 / モノオキシゲナーゼ / フラビン還元酵素 / 発光関連酵素 / X線結晶撞造解析 / ニトロ還元酵素 / 発光細菌 / 発光反応 / X線結晶構造解析

Research Abstract

発光細菌の発光系は発光反応を触媒するモノオキシゲナーゼであるルシフェラーゼと、そのルシフェラーゼに発光基質の還元型FMNと飽和長鎖脂肪族アルデヒドを供給する共役系酵素(FMN還元酵素FRaseI及び脂肪酸還元酵素)により構成される。Photobacterium(Vibrio)harveyiのルシフェラーゼとFMN還元酵素間の直接相互作用の証拠が見出されており、共役系を含めた複合体による効率の良い発光反応系は大変興味深い。Photorhabdus luminescensのルシフェラーゼがFMN還元酵素と比較的安定な複合体を形成することから、そのルシフェラーゼの大量発現、精製、結晶化を行った。現在、球晶が得られており、解析可能な単結晶を得るために条件検討を行っている。また、FMN還元酵素のFRaseIとアミノ酸配列上相同性が高いものの機能が明確に異なる酵素として大腸菌のニトロ還元酵素NfsBがある。大腸菌はニトロ還元酵素としてNfsBに加えてNfsAも有する。本研究では、酵素群の構造機能相関を原子レベルで詳細に解明するために、これらの酵素群のX線結晶構造解析を酵素反応速度解析とともに行なった。われわれは既に、FRaseI、NfsAおよびNfsB-NAD^+複合体の結晶構造解析をそれぞれ分解能1.8Å、1.7Åおよび2.0Åで行っている。さらに、Phe124をSer残基に置換したNfsBの変異体がフラビン還元酵素のような基質特異性を示すことから、NfsBのF124S変異体の結晶構造解析も行った。

Research Products

• [Publications] Kato, Y., Ito, M., Kawai, K., Nagata, K., Tanokura, M: "Determinants of ligand specificity in groups I and IV WW domains as studied by surface plasmon resonance and model building"Journal of Biological Chemistry. 277・12. 10173-10177 (2002)
• [Publications] Sawano, Y., Muramatsu, T., Hatano, K., Nagata, K., Tanokura, M: "Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform"Journal of Biological Chemistry. 277・31. 28222-28227 (2002)
• [Publications] Iwasaki, W., Sasaki, H., Nakamura, A., Kohama, K., Tanokura, M.: "Metal-free and Ca^<2+>-bound structures of a multidomain EF-hand protein, CBP40, from the lower eukaryote, Physarum polycephalum"Structure. 11・1. 75-85 (2003)
• [Publications] Hatano, K., Kojima, M., Tanokura, M., Takahashi, K: "Nuclear magnetic resonance studies on the pK_a values and interactions of ionizable groups in bromelain inhibitor VI from pineapple stem"Biological Chemistry. 384・1. 93-104 (2003)
• [Publications] Maeda, M., Takeuchi, K., Kojima, M., Tanokura, M., Kimura, K., Amemiya, Y., Kihara, H., Takahashi, K: "Kinetic studies of unfolding process of aspergillopepsinn II by pH-jump methods"Biochemical and Biophysical Research communications. 301・3. 745-750 (2003)
• [Publications] Katayama, H., Nagata, K., Ohira, T., YF., Tanokura, M., Nagasawa, H.: "The solution structure of molt-inhibiting hormone from the kuruma prawn Marsupenaeus japonicus"Journal of Biological Chemistry. (in press). (2003)
• [Publications] 田之倉優, 阿久津秀雄, 村松知成: "生化学キーノート(Hammes, B.D, Hooper, N.M.著)"シュプリンガーフェアラーク東京. 464 (2002)
• [Publications] 田之倉優, 村松知成, 八木澤 仁: "分子生物学キーノート(Turner, P.C., McLennan, A.G., Bates, A.D., White, M.R.H.著)"シュプリンガーフェアラーク東京. 398 (2002)
• [Publications] Sakai, N., Yao, M., Itou, H., Watanabe, N., Yumoto, F., Tanokura, M., Tanaka, I.: "The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshli OT3 in complex with Mg-ADP."Structure. 9・9. 817-826 (2001)
• [Publications] Yumoto, F., Nara, M., Kagi, H., Iwasaki, W., Ojiina, T., Nishita, K., Nagata, K., Tanokura, M.: "Coordination structures of Ca^<2+> and Mg^<2+> in Akazara scallop troponin C in solution : FTIR spectroscopy of side-chain COO^-Groups"European Journal of Biochemistry. 268. 6284-6290 (2001)
• [Publications] Tanaka, S., Ataha, M., Kubota, T., Soga, T., Homma, K., Lee, W.C., Tanokura, M.: "The effect of amphiphillic additives on the growth and morphology of Aspergillus niger acid proteinase A crystals"Journal of Crystal Growth. 234. 247-254 (2002)
• [Publications] Kato, Y., Ito, M., Kawai, K., Nagata, K., Tanokura, M.: "Determinants of ligand specificity in groups I and IV WW domains as studied by surface plasmon resonance and model building"Journal of Biological Chemistry. 277(in press). (2002)
• [Publications] Taka,H.,Kaga,N.,Fujimura,T.,Mineki,R.,Imaizumi,M.,Suzuki,Y.,Suzuki,R.,Tanokura,M.,Shindo,N.and Murayama,K: "Rapid determination of parvalbumin from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing and amino acid analysis."Journal of Biochemistry. 64(4). 852-857 (2000)
• [Publications] Suzuki,R.,Nagata,K.,Kawakami,M.,Nemoto,N.,Furutani,M.,Adachi,K.,Maruyama,T.and Tanokura,M.: "Assignment of ^1H, ^<13>C and ^<15>N resonances of FKBP from Methanococcus thermolithotrophicus."Journal of Biochemistry. 17(2). 183-184 (2000)
• [Publications] Murayama,K.,Shindo,N.,Suzuki.R.,Kawakami,M.,Mineki,R.,Taka,H.,Kazumo,S.,Nagata,K.,Maruyama,T.and Tanokura,M.: "Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence."Electrophoresis. 21. 1733-1739 (2000)
• [Publications] Onishi,H.,Konishi,K.,Fujiwara,K.,Hayakawa,K.,Tanokura,M.Martinez,H.M.and Morales,M.F.: "On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide."Proceedings of National Academy of Sciences of U.S.A.. 97(21). 11203-11208 (2000)
• [Publications] Nagata,K.,Kudo,N.,Abe,K.,Arai,S.and Tanokura,M.: "Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L.japonica"Biochemistry. 39. 14753-14760 (2000)
• [Publications] Kobori,T.,Sasaki,H.,Lee,W.G.,Zenno,S.,Saigo,K.,Murphy,M.E.P.and Tanokura,M.: "Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds. Alteration of pyridine nucleotide binding by a single amino acdi substitution."Journal of Biological Chemistry. 276(4). 2816-2823 (2001)
• [Publications] Iwasaki, W., Sasaki, H., Nakamura, A., Kohama, K. and Tanokura, M.: "Crystallization and preliminary X-ray diffraction studies of a 40kDA calcium binding protein specifically expressed in plasmodia of Physarum polycephlum"J. Biochem.. 126(1). 7-9 (1999)
• [Publications] Kobori, T., Lee, W.C., Akagi, T., Sasaki, H., Zenno, S., Saigo, K. and Tanokura, M: "Crystallization and preliminary crystallographic analysis of major nitroreductase from Escherichia coli"Acta Crystal.. D55. 1901-1902 (1999)
• [Publications] 田之倉優、小堀俊郎: "フラビン還元酵素の構造決定と機能解析"バイオサイエンスとバイオインダストリー. 56(5). 319-322 (1998)
• [Publications] 小堀俊郎、田之倉優: "フラビン還元酵素Fraselとそのファミリー"化学と生物. 36(6). 355-356 (1998)
• [Publications] 小堀俊郎、田之倉優: "発光細菌Vibrio fischeriのフラビン還元酵素の結晶構造"構造生物. 5(1). 9-14 (1999)
• [Publications] Lee, W.C., Hirai, T., Kobori, T., Sasaki, H., Koike, H., Tanokura, M., Zenno, S., Saigo, K., Nishino, T., Murphy, M.E.P., and Adman, E.T.: "Reaction and substrate recognition of the flavin reductase FRaseI from bioluminescent bacterium Vibrio fischer : Biochemistry and X-ray crystallography of FRaseI. Flavins and Flavoproteins 1999 (Ghisla S., Kroneck, P., P, and Sund, H. eds.)"Rudorf-Weber-Agency for Scientific Publications, Berlin, Germany. 709-708 (1999)