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Elucidation of quinone cofactor biogenesis in copper amine oxidase on the basis of proton structures and flexibility analysis

Research Project

Project/Area Number 19K05694
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 37010:Bio-related chemistry
Research InstitutionOsaka University

Principal Investigator

Okajima Toshihide  大阪大学, 産業科学研究所, 准教授 (10247968)

Co-Investigator(Kenkyū-buntansha) 村川 武志  大阪医科薬科大学, 医学部, 助教 (90445990)
Project Period (FY) 2019-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2020: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2019: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywords結晶構造解析 / 翻訳後修飾 / ゆらぎ / 補酵素 / 酵素 / 反応機構 / 銅アミン酸化酵素 / 金属イオン / 酸化反応 / 中性子構造解析
Outline of Research at the Start

本研究は、銅含有アミン酸化酵素の自己触媒的な補酵素トパキノン(TPQ)の生成反応を対象として、酵素触媒反応に果たす金属イオンの役割の一端を詳細に解明することを目的とする。中性子結晶構造解析と高分解能X線結晶解析によって、LMCT中間体のプロトン座標とゆらぎの情報を獲得し、速度論解析と計算化学による解析を組み合わせることによって、LMCT中間体形成機構の詳細を明らかにする。最終的に、銅含有アミン酸化酵素の活性中心をひとつの酸化的反応場として捉え、各種の芳香族化合物においてLMCT中間体を形成させ、それを分子状酸素で酸化する新規酸化酵素を構築することをめざす。

Outline of Final Research Achievements

Aim of this study is to elucidate roles of metal ions on self catalytic cofactor biogenesis in copper amine oxidase from Arthrobacter globiformis. This study experimentally clarified that ligand-metal charge transfer (LMCT)intermediate is formed on a TPQ-precursor tyrosine residue at the early step of the biogenesis reaction. Neutron crystallography determined the structures including proton coordinates, providing important information about the situation of the precursor Tyr residue when that is activated by metal binding.

Academic Significance and Societal Importance of the Research Achievements

銅アミン酸化酵素のTPQ形成反応において、分子全体のゆらぎの存在が重要であることが判明した。加えて、ligand-metal charge transfer(LMCT)中間体を含む各種の金属結合型反応中間体構造のX線結晶構造や中性子構造など、新規な構造情報を決定することができた。とりわけ、中性子構造は活性中心残基のプロトン座標について、実験的に明らかにすることに成功した。得られた結果は、銅アミン酸化酵素をベースとして、新規な酸化反応を引き起こす活性中心構造を作り出すために重要な情報となった。

Report

(4 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Research-status Report
  • 2019 Research-status Report
  • Research Products

    (25 results)

All 2022 2021 2020 2019 Other

All Journal Article (10 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 9 results,  Open Access: 6 results) Presentation (7 results) (of which Int'l Joint Research: 1 results,  Invited: 3 results) Remarks (8 results)

  • [Journal Article] Re-evaluation of protein neutron crystallography with and without X-ray/neutron joint refinement2022

    • Author(s)
      Murakawa Takeshi、Kurihara Kazuo、Adachi Motoyasu、Kusaka Katsuhiro、Tanizawa Katsuyuki、Okajima Toshihide
    • Journal Title

      IUCrJ

      Volume: 9 Issue: 3 Pages: 342-348

    • DOI

      10.1107/s2052252522003657

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase2021

    • Author(s)
      Murakawa Takeshi、Suzuki Mamoru、Arima Toshi、Sugahara Michihiro、Tanaka Tomoyuki、Tanaka Rie、Iwata So、Nango Eriko、Tono Kensuke、Hayashi Hideyuki、Fukui Kenji、Yano Takato、Tanizawa Katsuyuki、Okajima Toshihide
    • Journal Title

      Acta Crystallographica Section F Structural Biology Communications

      Volume: 77 Issue: 10 Pages: 356-363

    • DOI

      10.1107/s2053230x21008967

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Neutron Crystallography Reveals Unprecedented Active-site Structure in Copper Amine Oxidase2021

    • Author(s)
      Murakawa Takeshi、Kurihara Kazuo、Tamada Taro、Kusaka Katsuhiro、Adachi Motoyasu、Okajima Toshihide
    • Journal Title

      J-PARC MLF ANNUAL REPORT

      Volume: 12 Pages: 14-16

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] X-Ray and Neutron Joint Refinement Displays Shared Proton and Keto Form of Quinone Cofactor in Copper Amine Oxidase2021

    • Author(s)
      Murakawa Takeshi、Kurihara Kazuo、Kusaka Katsuhiro、Okajima Toshihide、Adachi Motoyasu
    • Journal Title

      Photon Factory Highlights 2020

      Volume: 6 Pages: 42-43

    • Related Report
      2021 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Unique protonation states of aspartate and topaquinone in the active site of copper amine oxidase2020

    • Author(s)
      Shoji Mitsuo、Murakawa Takeshi、Boero Mauro、Shigeta Yasuteru、Hayashi Hideyuki、Okajima Toshihide
    • Journal Title

      RSC Advances

      Volume: 10 Issue: 63 Pages: 38631-38639

    • DOI

      10.1039/d0ra06365g

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] In crystallo thermodynamic analysis of the catalytic reaction in bacterial copper amine oxidase2020

    • Author(s)
      Takeshi Murakawa, Seiki Baba,Toshihide Okajima
    • Journal Title

      SPring-8/SACLA Research Frontiers 2019

      Volume: 1 Pages: 20-21

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] 宙に浮く水素イオン?!-大型タンパク質の中性子結晶構造解析で見えた特異な世界-(4月28日、プレス発表)2020

    • Author(s)
      村川武志、栗原和男、庄司光男、柴崎千枝、角南智子、玉田太郎、矢野直峰、山田太郎、日下勝弘、鈴木 守、重田育照、黒木良太、林 秀行、矢野貴人、谷澤克行、安達基泰、岡島俊英
    • Journal Title

      J-PARC News

      Volume: 181 Pages: 2-2

    • Related Report
      2020 Research-status Report
  • [Journal Article] In crystallo thermodynamic analysis of the catalytic reaction in bacterial copper amine oxidase2020

    • Author(s)
      Takeshi Murakawa, Seiki Baba, Toshihide Okajima
    • Journal Title

      SPring-8/SACLA Research Frontiers

      Volume: 印刷中

    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Journal Article] Neutron crystallography of copper amine oxidase reveals keto/enolate interconversion of the quinone cofactor and unusual proton sharing2020

    • Author(s)
      Takeshi Murakawa, Kazuo Kurihara, Mitsuo Shoji, Chie Shibazaki, Tomoko Sunami, Taro Tamada, Naomine Yano, Taro Yamada, Katsuhiro Kusaka, Mamoru Suzuki, Yasuteru Shigeta, Ryota Kuroki, Hideyuki Hayashi, Takato Yano, Katsuyuki Tanizawa, Motoyasu Adachi, Toshihide Okajima
    • Journal Title

      Proceedings of the National Academy of Sciences

      Volume: 印刷中 Issue: 20 Pages: 10818-10824

    • DOI

      10.1073/pnas.1922538117

    • Related Report
      2019 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] In crystallo thermodynamic analysis of the cofactor conformational change induced during the catalytic reaction of copper amine oxidase2019

    • Author(s)
      村川武志,馬場清喜,岡島俊英
    • Journal Title

      生化学

      Volume: 91 Issue: 4 Pages: 565-571

    • DOI

      10.14952/SEIKAGAKU.2019.910565

    • NAID

      40022022855

    • ISSN
      0037-1017
    • Year and Date
      2019-08-25
    • Related Report
      2019 Research-status Report
    • Peer Reviewed
  • [Presentation] QM/MM法による銅アミン酸化酵素のプロトン化状態についての理論解析2020

    • Author(s)
      庄司 光男、村川 武志、重田 育照、林 秀行、岡島 俊英
    • Organizer
      第34回分子シミュレーション討論会
    • Related Report
      2020 Research-status Report
  • [Presentation] 銅アミン酸化酵素のプロトン化状態についてのQM/MM解析2020

    • Author(s)
      庄司 光男、村川 武志、重田 育照、林 秀行、岡島 俊英
    • Organizer
      量子生命科学会第2回大会
    • Related Report
      2020 Research-status Report
  • [Presentation] Neutron crystallography of copper amine oxidase: Novel protonated structure in the active site2020

    • Author(s)
      岡島俊英
    • Organizer
      錯体化学会第70回討論会
    • Related Report
      2020 Research-status Report
    • Invited
  • [Presentation] 銅アミン酸化酵素の高分解能中性子結晶構造と触媒反応機構2020

    • Author(s)
      岡島俊英
    • Organizer
      2020 年度iBIX-JAXA-KEK 物構研-QST 合同タンパク質研究会
    • Related Report
      2020 Research-status Report
    • Invited
  • [Presentation] 銅アミン酸化酵素のトパキノン補酵素生合成反応における中間体形成の分光学的解析・速度論的解析2020

    • Author(s)
      酒井克輝、佐々木康、村川武志、谷澤克行、岡島俊英
    • Organizer
      日本農芸化学会2020年度大会
    • Related Report
      2019 Research-status Report
  • [Presentation] Structural basis for conformational change of the topaquinone cofactor during the catalytic reaction of bacterial copper amine oxidase2019

    • Author(s)
      Toshihide Okajima, Takeshi Murakawa, Seiki Baba, Satoshi Kanagawa, Hideyuki Hayashi, Takato Yano, Takashi Kumasaka, and Katsuyuki Tanizawa
    • Organizer
      33rd Annual Symposium of The Protein Society
    • Related Report
      2019 Research-status Report
    • Int'l Joint Research
  • [Presentation] 銅含有アミン酸化酵素触媒機構の“in crystallo” 熱力学的解析2019

    • Author(s)
      村川武志
    • Organizer
      第42回 SPring-8先端利用技術ワークショップ/大阪大学蛋白質研究所セミナー
    • Related Report
      2019 Research-status Report
    • Invited
  • [Remarks] 銅アミン酸化酵素の中性子結晶構造解析に成功 ―宙に浮いているようにみえる水素イオンなど初めて解明

    • URL

      https://www.ibaraki.ac.jp/news/2020/04/28010784.html

    • Related Report
      2020 Research-status Report
  • [Remarks] 宙に浮く水素イオン?! ―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      https://resou.osaka-u.ac.jp/ja/research/2020/20200428_1

    • Related Report
      2020 Research-status Report
  • [Remarks] 宙に浮く水素イオン?! ―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      https://www.sanken.osaka-u.ac.jp/hot_topics/topics_20200428/

    • Related Report
      2020 Research-status Report
  • [Remarks] 宙に浮く水素イオン?!―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      https://www.ccs.tsukuba.ac.jp/release-20200428/

    • Related Report
      2020 Research-status Report
  • [Remarks] 宙に浮く水素イオン?!―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      http://www.tsukuba.ac.jp/attention-research/p202004280400.html

    • Related Report
      2020 Research-status Report
  • [Remarks] 宙に浮く水素イオン?! 大型タンパク質の中性子結晶構造解析で見えた特異な世界

    • URL

      https://resou.osaka-u.ac.jp/ja/research/2020/20200428_1

    • Related Report
      2019 Research-status Report
  • [Remarks] 宙に浮く水素イオン?! ―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      https://www.sanken.osaka-u.ac.jp/hot_topics/topics_20200428/

    • Related Report
      2019 Research-status Report
  • [Remarks] 宙に浮く水素イオン?! ―大型タンパク質の中性子結晶構造解析で見えた特異な世界―

    • URL

      https://www.osaka-med.ac.jp/news/research/f2pjgc000000fjnt.html

    • Related Report
      2019 Research-status Report

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Published: 2019-04-18   Modified: 2023-01-30  

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