Identification and Functional Analysis of Stimuli-responsive Chemical Species during Enzyme Reaction

• Project Area: Stimuli-responsive Chemical Species for the Creation of Functional Molecules
• Project/Area Number: 24109017
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Science and Engineering
• Research Institution: Osaka University
• Principal Investigator: Inoue Tsuyohi 大阪大学, 工学研究科, 教授 (20263204)
• Co-Investigator(Kenkyū-buntansha): 中村 努 国立研究開発法人産業技術総合研究所, その他部局等, 研究員 (10357668) ; 松村 浩由 立命館大学, 生命科学部, 教授 (30324809)
• Project Period (FY): 2012-06-28 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥84,240,000 (Direct Cost: ¥64,800,000、Indirect Cost: ¥19,440,000); Fiscal Year 2016: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2015: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2014: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2013: ¥9,360,000 (Direct Cost: ¥7,200,000、Indirect Cost: ¥2,160,000); Fiscal Year 2012: ¥46,800,000 (Direct Cost: ¥36,000,000、Indirect Cost: ¥10,800,000)
• Keywords: チオレドキシンペルオキシダーゼ / 還元反応 / 酵素反応 / 反応中間体 / Ⅹ 線構造解析 / 中性子線解析 / Ⅹ線自由電子レーザー / SACLA / Ⅹ線構造解析 / 蛋白質 / X線 / ペルオキシレドキシン / スーパーオキシドディスムターゼ / 反応機構解明 / 中性子線構造解析

Outline of Final Research Achievements

Peroxiredoxin (Prx) has a Cys residue at its active site and shows the enzyme activity via S-OH intermediate containing hypervalent sulfur in the process of reducing hydrogen peroxide to water. For uncovering the presence of H atoms on the S atom by neutron analysis, we tried to obtain large crystals of Prx (ApPrx, PhPrx, TkPrx) derived from three types of hyperthermophilic archaea. None of them diffracted up to 3.0A resolution, and the presence of H atom on the S atom was not proved to date. However, the similar structure of S-OH intermediate existed in PhPrx, demonstrating the generality of the S-OH intermediate.

On the other hand, X-ray free electron laser is superior as a method to investigate stimuli-responsive chemical species. We have acquired a knowledge on structural change accompanying reduction upon X-ray irradiation was obtained from the completely oxidized form of nitrite reductase (NIR).

Research Products

• [Journal Article] A RuBisCO-mediated carbon metabolic pathway in methanogenic archaea. (2017)
  • Author(s): T. Kono, S. Mehrotra, C. Endo, N. Kizu, M. Matsuda, H. Kimura, E. Mizohata, T. Inoue, T. Hasunuma, A. Yokota, H. Matsumura, H. Ashida
  • Journal Title: Nat Commun Volume: 8 Pages: 14007-14007
  • DOI: 10.1038/ncomms14007
  • NAID: 120005972976
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Protein crystallization with paper (2016)
  • Author(s): Miki Matsuoka, Keisuke Kakinouchi, Hiroaki Adachi, Mihoko Maruyama, Shigeru Sugiyama, Satoshi Sano, Hiroshi Y. Yoshikawa, Yoshinori Takahashi, Masashi Yoshimura, Hiroyoshi Matsumura, Satoshi Murakami, Tsuyoshi Inoue, Yusuke Mori and Kazufumi Takano
  • Journal Title: Jpn. J. Appl. Phys., Volume: 55 Pages: 050302-050302
  • NAID: 210000146451
  • Peer Reviewed / Open Access
• [Journal Article] Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography (2016)
  • Author(s): Fukuda Y, Tse KM, Suzuki M, Diederichs K, Hirata K, Nakane T, Sugahara M, Nango E, Tono K, Joti Y, Kameshima T, Song C, Hatsui T, Yabashi M, Nureki O, Matsumura H, Inoue T, Iwata S, Mizohata E.
  • Journal Title: Proc. Natl. Acad. Sci. USA Volume: 未定 Pages: 2928-2933
  • DOI: 10.1093/jb/mvv133
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] A crystallization technique for obtaining large protein crystals with increased mechanical stability using agarose gel combined with a stirring technique (2016)
  • Author(s): M. Maruyama, Y. Hayashi, H. Y. Yoshikawa, S. Okada, H. Koizumi, M. Tachibana, S. Sugiyama, H. Adachi, H. Matsumura, T. Inoue, K. Takano, S. Murakami, M. Yoshimura, Y. Mori
  • Journal Title: Journal of Crystal Growth Volume: 452 Pages: 172-178
  • DOI: 10.1016/j.jcrysgro.2015.11.008
  • Peer Reviewed / Open Access
• [Journal Article] Promotion of protein crystal growth by actively switching crystal growth mode via femtosecond laser ablation (2016)
  • Author(s): Y. Tominaga, M. Maruyama, M. Yoshimura, H. Koizumi, M. Tachibana, S. Sugiyama, H. Adachi, K. Tsukamoto, H. Matsumura, K. Takano, S. Murakami, T. Inoue, H. Y. Yoshikawa, Y. Mori
  • Journal Title: NATURE PHOTONICS Volume: 10 Pages: 723-726
  • DOI: 10.1038/nphoton.2016.202
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Epiregulin recognition mechanisms by anti-epiregulin antibody 9E5: Structural, functional and molecular dynamics simulation analyses (2016)
  • Author(s): Kado Y1, Mizohata E2, Nagatoishi S3, Iijima M4, Shinoda K4, Miyafusa T3, Nakayama T2, Yoshizumi T2, Sugiyama A4, Kawamura T4, Lee YH4, Matsumura H2, Doi H4, Fujitani H4, Kodama T4, Shibasaki Y4, Tsumoto K5, Inoue T6.
  • Journal Title: J. Biol. Chem. Volume: 291 Pages: 2319-2330
  • DOI: 10.1074/jbc.m115.656009
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Redox-coupled structural changes in nitrite reductase revealed by serial femtosecond and microfocus crystallography (2016)
  • Author(s): Fukuda Y, Tse KM, Nakane T, Nakatsu T, Suzuki M, Sugahara M, Inoue S, Masuda T, Yumoto F, Matsugaki N, Nango E, Tono K, Joti Y, Kameshima T, Song C, Hatsui T, Yabashi M, Nureki O, Murphy ME, Inoue T, Iwata S, Mizohata E.
  • Journal Title: Journal of biochemistry Volume: 113 Pages: 2828-2933
  • DOI: 10.1073/pnas.1517770113
  • NAID: 40020833586
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Insights into unknown foreign ligand in copper nitrite reductase (2015)
  • Author(s): Yohta Fukuda, Tse Ka Man, Yuji Kado, Eiichi Mizohata, Hiroyoshi Matsumura, Tsuyoshi Inoue
  • Journal Title: Biochem. Biophys. Res. Commun., Volume: 464（２） Pages: 622-628
  • DOI: 10.1016/j.bbrc.2015.07.025
  • Peer Reviewed / Open Access
• [Journal Article] Structure-based design and synthesis of a bivalent iminobiotin analogue showing strong affinity toward a low immunogenic streptavidin mutant (2015)
  • Author(s): Kawato T, Mizohata E, Shimizu Y, Meshizuka T, Yamamoto T, Takasu N, Matsuoka M, Matsumura H, Kodama T, Kanai M, Doi H, Inoue T, Sugiyama A
  • Journal Title: Biosci. Biotechnol. Biochem. Volume: 79 Pages: 640-642
  • DOI: 10.1080/09168451.2014.991692
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Grease matrix as a versatile carrier of proteins for serial crystallography. (2015)
  • Author(s): Sugahara, M.; Mizohata, E.; Nango, E.; Suzuki, M.; Tanaka, T.; Masuda, T.; Tanaka, R.; Shimamura, T.; Tanaka, Y.; Suno, C.; Ihara, K.; Pan, D.; Kakinouchi, K.; Sugiyama, S.; Murata, M.; Inoue, T.; Tono, K.; Song, C.; Park, J.; Kameshima, T.; Hatsui, T.; Joti, Y.; Yabashi, M.; Iwata, S
  • Journal Title: Nature Methods, Volume: 12 Pages: 61-63
  • NAID: 120005682565
  • Peer Reviewed / Open Access
• [Journal Article] Structure-based design of a streptavidin mutant specific for an artificial biotin analogue (2015)
  • Author(s): Kawato, T.; Mizohata, E.; Shimizu, Y.; Meshizuka, T.; Yamamoto, T. Takasu, N.; Matsuoka, M.; Matsumura, H.; Kodama, T.; Kanai, M.; Doi, H.; Inoue, T.; Sugiyama, A.
  • Journal Title: J. Biochem. Volume: 1 Pages: 467-475
  • DOI: 10.1093/jb/mvv004
  • Peer Reviewed / Open Access
• [Journal Article] Crystal structure of streptavidin mutant with low immunogenicity (2015)
  • Author(s): Kawato T, Mizohata E, Meshizuka T, Doi H, Kawamura T, Matsumura H, Yumura K, Tsumoto K, Kodama T, Inoue T & Sugiyama A.
  • Journal Title: Journal of Bioscience and Bioengineering Volume: 119(6) Issue: 6 Pages: 642-647
  • DOI: 10.1016/j.jbiosc.2014.10.025
  • NAID: 110009970751
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Structural features of interfacial tyrosine residue in ROBO1 fibronectin domain-antibody complex: Crystallographic, thermodynamic, and molecular dynamic analyses. (2014)
  • Author(s): Nakayama, T.; Mizohata, E.; Yamashita, T.; Nagatoishi, S.; Nakakido, M.; Iwanari, H.; Mochizuki, Y.; Kado, Y.; Yokota, Y.; Satoh, R.; Tsumoto, K.; Fujitani, H.; Kodama, T.; Hamakubo, T.; Inoue T
  • Journal Title: Protein Science, Volume: 24 Pages: 328-340
  • Peer Reviewed / Open Access
• [Journal Article] pH-controlled Degradation and Thermal Stability of a Porous Poly(γ-glutamic acid) Monolith Crosslinked with an Oxazoline-functionalized Polymer (2014)
  • Author(s): S. B. Park, T Fujimoto, E Mizohata, T Inoue, M.- H. Sung, and H. Uyama
  • Journal Title: Polym. Degrad. Stab. Volume: 23 Pages: 99-104
  • DOI: 10.1016/j.polymdegradstab.2013.11.019
  • Peer Reviewed
• [Journal Article] Structural insights into the function of a thermostable copper-containing nitrite reductase (2014)
  • Author(s): Y Fukuda, et al.
  • Journal Title: J. Biochem. Volume: 155 Pages: 123-135
  • DOI: 10.1093/jb/mvt107
  • Peer Reviewed
• [Journal Article] Jumonji/Arid1b (Jarid1b) protein modulates human esophageal cancer cell growth (2013)
  • Author(s): Kano, Y., Konno, M., Ohta, K., Haraguchi, N., Nishikawa, S., Kagawa, Y., Hamabe, A., Hasegawa, S., Ogawa, H., Fukusumi, T., Noguchi, Y., Ozaki, M., Kudo, T., Sakai, D., Satoh, T., Ishii, M., Mizohata, E., Inoue, T., Mori, M., Doki, Y., Ishii, H.
  • Journal Title: Molecular and Clinical Oncology Volume: 1 Pages: 753-757
  • DOI: 10.3892/mco.2013.127
  • Peer Reviewed
• [Journal Article] Structure of peroxiredoxin from anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (2013)
  • Author(s): T. Nakamura*, A. Mori, M. Niiyama, H. Matsumura, C. Tokuyama, J. Morita, K. Uegaki and T. Inoue
  • Journal Title: Acta. Cryst. F Volume: F69 Pages: 719-722
  • DOI: 10.1107/s1744309113014036
  • Peer Reviewed
• [Journal Article] Expression, purification, crystallization and preliminary crystallographic analysis of spermidine acetyltransferase from Escherichia coli (2013)
  • Author(s): Niiyama, M. et al.
  • Journal Title: Acta Crystallogr Sect F Struct Biol Cryst Commun Volume: 69 Pages: 884-887
  • DOI: 10.1107/s1744309113017132
  • Peer Reviewed
• [Journal Article] Expression, purification, crystallization and preliminary crystallographic study of FtsA from methicillin-resistant Staphylococcus aureus (2013)
  • Author(s): J. Fujita, Y. Miyazaki, M. Hirose, C. Nagao, E. Mizohata, Y. Matsumoto, K. Mizuguchi, T. Inoue, and H. Matsumura
  • Journal Title: Acta. Cryst. F Volume: F69 Pages: 895-898
  • DOI: 10.1107/s1744309113017727
  • Peer Reviewed
• [Journal Article] Targeting BIG3-PHB2 interaction to overcome tamoxifen resistance in breast cancer cells. (2013)
  • Author(s): T. Yoshimaru, M. Komatsu, T. Matsuo, Y.-A. Chen, Y. Murakami, K. Mizuguchi, E. Mizohata, T. Inoue, M. Akiyama, R. Yamaguchi, S. Imoto, S. Miyano, Y. Miyoshi, M. Sasa, Y. Nakamura, and T. Katagiri
  • Journal Title: Nature communications Volume: 4 Pages: 2433-2433
  • DOI: 10.1038/ncomms3443
  • Peer Reviewed
• [Journal Article] Construction and characterization of functional anti-epiregulin humanized monoclonal antibodies (2013)
  • Author(s): Y.-H. Lee, M. Iijima, Y. Kado, T. Inoue, A. Sugiyama, H. Doi, Y. Shibasaki, T. Kodama
  • Journal Title: Biochem. Biophys. Res. Commun Volume: 441 Pages: 1011-1017
  • DOI: 10.1016/j.bbrc.2013.11.014
  • Peer Reviewed
• [Journal Article] Structure of the human-heart fatty-acid-binding protein 3 in complex with the fluorescent probe 1-anilinonaphthalene-8-sulphonic acid (2013)
  • Author(s): M. Hirose, S. Sugiyama, M. Niiyama, D. Matsuoka, T. Hara, E. Mizohata, S. Murakami, T. Inoue, S. Matsuoka, and M. Murata
  • Journal Title: 10.1107/S0909049513021298 Volume: 20 Pages: 923-928
  • Peer Reviewed
• [Journal Article] C(sp(3))-H Bond Hydroxylation Catalyzed by Myoglobin Reconstituted with Manganese Porphycene (2013)
  • Author(s): Koji Oohora, Yushi Kihira, Eiichi Mizohata, Tsuyoshi Inoue, Takashi Hayashi
  • Journal Title: J. Am. Chem. Soc. Volume: 135 Pages: 17282-17285
  • DOI: 10.1021/ja409404k
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Crystal structure of rice Rubisco and implications for activation induced by positive effectors NADPH and 6-phosphogluconate (2012)
  • Author(s): Hiroyoshi Matsumura
  • Journal Title: J. Mol .Biol. Volume: 422(1) Pages: 75-86
  • DOI: 10.1016/j.jmb.2012.05.014
  • Peer Reviewed
• [Journal Article] Spatially precise, soft microseeding of single protein crystals by femtosecond laser ablation (2012)
  • Author(s): Hiroshi Yoshikawa
  • Journal Title: Crystal Growth Design Volume: 12(9) Pages: 4334-4339
  • DOI: 10.1021/cg300018t
  • Peer Reviewed
• [Presentation] The molecular design of a mutant streptavidin with high specificity for only an artificial biotin derivative (2017)
  • Author(s): Tsuyoshi Inoue, Tatsuya Kawato, Eiichi Mizohata, Yohei Shimizu, Tomohiro Meshizuka, Tomohiro Yamamoto, Noriaki Takasu, Masahiro Matsuoka, Tatsuhiko Kodama, Motomu Kanai, Hirofumi Doi, Akira Sugiyama
  • Organizer: 11th Pure and Applied Chemistry International Conference 2017 (PACCON 2017)
  • Place of Presentation: CENTRA GOVERNMENT COMPLEX HOTEL & CONVENTION CENTRE CHAENG WATTHANA, Bangkok
  • Year and Date: 2017-02-02
  • Int'l Joint Research / Invited
• [Presentation] Structural Bioinorganic Chemistry of Copper Enzymes (2016)
  • Author(s): Yohta Fukuda, Eiichi Mizohata, Tsuyoshi Inoue
  • Organizer: The 4th International Symposium for Young Chemists on Stimuli-Responsive Chemical Species for the Creation of Functional Molecules
  • Place of Presentation: 大阪大学吹田キャンパス 工学研究科 サントリーメモリアルホール（大阪府・吹田市）
  • Year and Date: 2016-12-12
  • Int'l Joint Research / Invited
• [Presentation] 中性子用大型良質タンパク質結晶育成を目指して (2016)
  • Author(s): 安達宏昭、岡田詩乃、杉山成、丸山美帆子、高野和文、吉川洋史、村上聡、松村浩由、井上豪、森勇介
  • Organizer: 日本結晶学会平成28年度年会
  • Place of Presentation: 茨城県立県民文化センター（茨城県・水戸市）
  • Year and Date: 2016-11-17
• [Presentation] SACLA が解明した銅含有亜硝酸還元酵素の常温無損傷構造 (2016)
  • Author(s): 溝端 栄一
  • Organizer: 第16回日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場（福岡県・福岡市）
  • Year and Date: 2016-06-07
• [Presentation] エピレギュリン－抗体複合体構造解析に基づく分子認識機構の解明 (2016)
  • Author(s): 門 祐示、溝端 栄一、長門石 曉、 飯嶋 真理子、篠田 恵子、宮房 孝光、 中山 泰亮、吉住 拓真、杉山 暁、 川村 猛、Lee Yougn-Hun、松村 浩由 、土居 洋文、児玉 龍彦、柴崎 芳一、津本 浩平、井上 豪
  • Organizer: 第16回日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場（福岡県・福岡市）
  • Year and Date: 2016-06-07
• [Presentation] 親和性向上を目的とした抗ROBO1 scFv の変異体解析 (2016)
  • Author(s): 由井 杏奈、工藤 翔太、秋葉 宏樹、中木戸 誠、長門石 曉、井上 豪、浜窪 隆雄、津本 浩平
  • Organizer: 第16回日本蛋白質科学会年会
  • Place of Presentation: 福岡国際会議場（福岡県・福岡市）
  • Year and Date: 2016-06-07
• [Presentation] Growth of High-Quality and Large Protein Crystals for Neutron Crystallography (2013)
  • Author(s): Hiroyoshi Matsumura
  • Organizer: 感応性化学種が拓く新物質科学 第１回国際シンポジウム
  • Place of Presentation: メルパルク広島
  • Year and Date: 2013-03-26
• [Presentation] Hypervalent Intermediate Observed during Oxidation of Peroxiredoxin from an Aerobic Hyperthermophilic Archaeon Aeropyrum pernix K1 (2013)
  • Author(s): Tsuyoshi Inoue
  • Organizer: 感応性化学種が拓く新物質科学 第１回国際シンポジウム
  • Place of Presentation: メルパルク広島
  • Year and Date: 2013-03-26
• [Presentation] Crystal structure of Archaeal Peroxiredoxin from an Anaerobic Hyperthermophilic Archaeon Pyrococcus horikoshii (2013)
  • Author(s): Tsutomu Nakamura
  • Organizer: 感応性化学種が拓く新物質科学 第１回国際シンポジウム
  • Place of Presentation: メルパルク広島
  • Year and Date: 2013-03-26
• [Presentation] 嫌気性古細菌由来ペルオキシレドキシンの結晶化と構造解析 (2013)
  • Author(s): 中村 努
  • Organizer: 第12 回 産総研・産技連LS-BT 合同研究発表会
  • Place of Presentation: 産総研つくばセンター
  • Year and Date: 2013-02-05
• [Presentation] 生体酵素系に生成する感応性化学種の同定と機能解明 (2012)
  • Author(s): 井上 豪
  • Organizer: 感応性化学種が拓く新物質科学 第１回公開シンポジウム
  • Place of Presentation: 京都大学・宇治おうばくプラザ・きはだホール
• [Remarks] 井上豪Home Page
  • URL: http://www.chem.eng.osaka-u.ac.jp/~inoue-tken/ja/staffs/inoue/index.html
• [Remarks] Hiroyoshi Matsumura (松村 浩由) Homepage
  • URL: http://www.chem.eng.osaka-u.ac.jp/~inoue-tken/ja/staffs/matsumura/
• [Remarks] 中村努Home Page
  • URL: http://unit.aist.go.jp/hri/group/bmd-rg/member/nakamura.htm