• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Structure analysis of monooxygenase using XFEL and microfluidic device

Research Project

Project/Area Number 19K22403
Research Category

Grant-in-Aid for Challenging Research (Exploratory)

Allocation TypeMulti-year Fund
Review Section Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
Research InstitutionInstitute of Physical and Chemical Research

Principal Investigator

Sugimoto Hiroshi  国立研究開発法人理化学研究所, 放射光科学研究センター, 専任研究員 (90344043)

Co-Investigator(Kenkyū-buntansha) 真栄城 正寿  北海道大学, 工学研究院, 助教 (40744248)
Project Period (FY) 2019-06-28 – 2021-03-31
Project Status Completed (Fiscal Year 2020)
Budget Amount *help
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2020: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2019: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Keywordsヘム / X線結晶構造解析 / X線自由電子レーザー / XFEL / X線 / X線回折
Outline of Research at the Start

モノオキシゲナーゼ(一原子酸素添加酵素)であるシトクロムP450ファミリーのタンパク質は、ヘムを補欠因子として様々な基質を酸化して生命活動の維持を担っている。これまでの研究からは、いったん基質がタンパク質の基質ポケットに結合した後に、ヘム鉄の酸化状態や酸素リガンドの化学状態の変化に伴って、基質が活性中心の鉄イオン側へ大きく引き寄せられる機構が推測されてきた。本研究は、このような酵素の触媒反応における動的な分子メカニズムの解明を目的としており、X線自由電子レーザーによる測定と最先端のマイクロ流体技術、そして分光技術を駆使した新しい構造解析の確立に挑戦する。

Outline of Final Research Achievements

Monooxygenase reactions catalyzed by cytochrome P450 family require the sequential input of two reducing equivalents (i.e., two electrons and two protons) which activate the oxygen molecule. We developed a prototype of device to control such a multi-step reaction of monooxygenase in crystalline phase. We performed a feasibility study of X-ray diffraction experiments using this device with microcrystals of cytochrome P450 at X-ray free electron laser (XFEL) facility. The result has shown that the device is useful for data collection system of serial crystallography using XFEL and photo-caged reagents, which pave the way to evaluate the dynamic mechanism of proteins by time-resolved crystallography even if their function would not contain a photoreaction.

Academic Significance and Societal Importance of the Research Achievements

ヒトのシトクロムP450はホルモン合成や薬物代謝に深く関与することから、疾病を防ぐための研究が長年にわたって行われてきた。また、植物や細菌など多くの生物にもシトクロムP450は存在し、植物の農薬への耐性や環境汚染物質の浄化の研究などに用いられている。したがって、本課題が実施したP450の活性部位での多様な基質認識や反応メカニズムの研究は、人間にとって有用な化合物生産のための触媒としてシトクロムP450の反応を利用するための研究開発へと発展すると期待される。

Report

(3 results)
  • 2020 Annual Research Report   Final Research Report ( PDF )
  • 2019 Research-status Report
  • Research Products

    (10 results)

All 2020 2019 Other

All Int'l Joint Research (2 results) Journal Article (6 results) (of which Int'l Joint Research: 3 results,  Peer Reviewed: 6 results,  Open Access: 5 results) Presentation (2 results)

  • [Int'l Joint Research] University of Essex/Diamond Light Source(英国)

    • Related Report
      2020 Annual Research Report
  • [Int'l Joint Research] University of Essex/Diamond Light Source(英国)

    • Related Report
      2019 Research-status Report
  • [Journal Article] Serial Femtosecond Zero Dose Crystallography Captures a Water‐Free Distal Heme Site in a Dye‐Decolorising Peroxidase to Reveal a Catalytic Role for an Arginine in Fe IV =O Formation2020

    • Author(s)
      Lucic Marina、Svistunenko Dimitri A.、Wilson Michael T.、Chaplin Amanda K.、Davy Bradley、Ebrahim Ali、Axford Danny、Tosha Takehiko、Sugimoto Hiroshi、Owada Shigeki、Dworkowski Florian S. N.、Tews Ivo、Owen Robin L.、Hough Michael A.、Worrall Jonathan A. R.
    • Journal Title

      Angewandte Chemie International Edition

      Volume: 59 Issue: 48 Pages: 21656-21662

    • DOI

      10.1002/anie.202008622

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Room-temperature crystallography using a microfluidic protein crystal array device and its application to protein-ligand complex structure analysis2020

    • Author(s)
      Maeki Masatoshi、Ito Sho、Takeda Reo、Ueno Go、Ishida Akihiko、Tani Hirofumi、Yamamoto Masaki、Tokeshi Manabu
    • Journal Title

      Chemical Science

      Volume: 11 Issue: 34 Pages: 9072-9087

    • DOI

      10.1039/d0sc02117b

    • Related Report
      2020 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Crystals in minutes: instant on-site microcrystallisation of various flavours of the CYP102A1 (P450BM3) haem domain2020

    • Author(s)
      Stanfield, J. K., Omura, K., Matsumoto, A., Kasai, C., Sugimoto, H., Shiro, Y., Watanabe, Y., Shoji, O.
    • Journal Title

      Angew. Chem. Int. Ed.

      Volume: 59 Issue: 19 Pages: 7611-7618

    • DOI

      10.1002/anie.201913407

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA2020

    • Author(s)
      Suga Michihiro、Shimada Atsuhiro、Akita Fusamichi、Shen Jian-Ren、Tosha Takehiko、Sugimoto Hiroshi
    • Journal Title

      Biochimica et Biophysica Acta (BBA) - General Subjects

      Volume: 1864 Issue: 2 Pages: 129466-129466

    • DOI

      10.1016/j.bbagen.2019.129466

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] High-throughput structures of protein-ligand complexes at room temperature using serial femtosecond crystallography2019

    • Author(s)
      T. Moreno-Chicano, A. Ebrahim, D. Axford, M. V. Appleby, J. H. Beale, AMoreno-Chicano, T., Ebrahim, A., Axford, D., Appleby, M. V., Beale, J. H., Chaplin, A. K., Duyvesteyn, H. M. E., Ghiladi, R. A., Owada, S., Sherrell, D. A., Strange, R. W., Sugimoto, H., Tono, K., Worrall, J. A. R., Owen, R. L., Hough, M. A.
    • Journal Title

      IUCrJ

      Volume: 6 Issue: 6 Pages: 1074-1074

    • DOI

      10.1107/s2052252519011655

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Int'l Joint Research
  • [Journal Article] Dose-resolved serial synchrotron and XFEL structures of radiation sensitive metalloproteins2019

    • Author(s)
      Ebrahim, A., Moreno-Chicano, T., Appleby, M. V., Chaplin, A. K., Beale, J. H., Sherrell, D. A., Duyvesteyn, H. M. E., Owada, S., Tono, K., Sugimoto, H., Strange, R. W., Worrall, J. A. R., Axford, D., Owen, R. L., Hough, M. A.
    • Journal Title

      IUCrJ

      Volume: 6 Issue: 4 Pages: 543-543

    • DOI

      10.1107/s2052252519003956

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] マイクロデバイスを用いたタンパク質結晶の室温複合体構造解析2020

    • Author(s)
      竹田怜央, 真栄城正寿, 伊藤翔, 上野剛, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学
    • Organizer
      化学とマイクロナノシステム学会 第42回研究会(CHEMINAS42)
    • Related Report
      2020 Annual Research Report
  • [Presentation] 常温X線結晶構造解析のためのマイクロデバイスの開発2020

    • Author(s)
      舟久保智瑛, 真栄城正寿, 伊藤翔, 上野剛, 石田晃彦, 谷博文, 山本雅貴, 渡慶次学
    • Organizer
      第80回分析化学討論会
    • Related Report
      2020 Annual Research Report

URL: 

Published: 2019-07-04   Modified: 2022-01-27  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi