Reaction mechanism study of heme containing dioxygenase by stopped-flow resonance Raman spectroscopy

• Project/Area Number: 19K05698
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 37010:Bio-related chemistry
• Research Institution: University of Hyogo
• Principal Investigator: Yanagisawa Sachiko 兵庫県立大学, 理学研究科, 准教授 (60557982)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000); Fiscal Year 2021: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: ヘム酵素 / 二原子酸素添加酵素 / ストップトフロー / 共鳴ラマン / 免疫抑制 / 2原子酸素添加酵素 / stopped-flow / 共鳴ラマン分光法 / 可視紫外吸収分光法 / 紫外可視吸収分光法 / ２原子酸素添加酵素 / ヘムタンパク質 / ジオキシゲナーゼ / ストップトフローラマン

Outline of Research at the Start

ヒトインドールアミン2,3ジオキシゲナーゼ1(IDO)は, ヘム酵素として唯一の2原子酸素添加反応である, トリプトファン(Trp)主要代謝経路の最初の反応を触媒する誘導タンパク質である. この酸素添加反応は収支として電子とプロトンを必要とせず, ヘム酵素として特異な反応で, 反応機構全容は不明である. 本研究では, ストップトフロー共鳴ラマン分光法を用い, これまでは検出ができなかった‘反応の一部始終’を, 振動スペクトルとして検出することにより, IDO反応機構全容の解明を目指す.

Outline of Final Research Achievements

Indoleamine 2,3-dioxygenase, which is a heme conatining enzyme, incorporates two oxygen atoms into Tryptophan. This reaction is the only dioxygenase reaction catalized by heme enzyme. On the other hand, this reaction is involved in immune suppression, and thus IDO is a immunotherapeutic target. However, the reaction mechanism of IDO is not yet fully understood. In this study, we monitored a whole enzymatic reaction by spectroscopy and asigned the structure and state of reaction intermediates to understand the reaction mechanism. As a result, a presence of two different types of ternary complex intermediates and the importance of a high reactivity of Trp are suggested.

Academic Significance and Societal Importance of the Research Achievements

IDOは免疫療法のターゲットであり、治療薬としてのIDO阻害剤の探索が盛んに行われている。本研究では、既報の中間体が２状態存在し時間経過に伴って増減する様子を捉え、また、この中間体の安定化の原因を示唆した。中間体が安定に存在することは、活性を低下させる要因であり、阻害剤をデザインする上で有益な情報といえる。同時に、ヘム酵素による唯一のニ原子酸素添加反応の反応機構解明の鍵となる、未検出中間体を検出するための実験をデザインする上でも有益な情報である。

Research Products

• [Journal Article] A synthetic model for the possible FeIV2(μ-O)2 core of methane monooxygenase intermediate Q derived from a structurally characterized FeIIIFeIV(μ-O)2 complex. (2022)
  • Author(s): Mikata, Y., Aono, Y., Yamamoto, C., Nakayama, H., Matsumoto, A., Kotegawa, F., Harada, M., Katano, H., Kobayashi, Y., Yanagisawa, S., Kubo, M., Kajiwara, A., Kodera, M
  • Journal Title: Inorganic Chemistry Volume: 61 Issue: 2 Pages: 786-790
  • DOI: 10.1021/acs.inorgchem.1c02699
  • Peer Reviewed
• [Journal Article] Revisiting Alkane Hydroxylation with m‐CPBA (mChloroperbenzoic Acid) Catalyzed by Nickel(II) Complexes (2021)
  • Author(s): Shinke, T., Itoh, M., Wada, T., Morimoto, Y., Yanagisawa, S., Sugimoto, H., Kubo, M., Itoh S.
  • Journal Title: Chemistry A European Journal Volume: 27 Issue: 59 Pages: 14730-14737
  • DOI: 10.1002/chem.202102532
  • Peer Reviewed
• [Journal Article] Short-lived intermediate in N2O generation by P450 NO reductase captured by time-resolved IR spectroscopy and XFEL crystallography (2021)
  • Author(s): Nomura, T., Kimura, T., Kanematsu, Y., Yamada, D., Yamashita, K., Hirata, K., Ueno, G., Murakami, H., Hisano, T., Yamagiwa, R., Takeda, H., Gopalasingam, C., Kousaka, R., Yanagisawa, S., Shoji, O., Kumasaka, T., Yamamoto, M., Takano, Y., Sugimoto, H., Tosha, T., Kubo, M., Shiro, Y
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 118 Issue: 21
  • DOI: 10.1073/pnas.2101481118
  • Peer Reviewed / Open Access
• [Journal Article] Effect of the Electron Density of the Heme Fe Atom on the Nature of Fe-O2 Bonding in Oxy Myoglobin (2020)
  • Author(s): Yamamoto Yasuhiko、Hasegawa Kazuyasu、Shibata Tomokazu、Momotake Atsuya、Ogura Takashi、Yanagisawa Sachiko、Neya Saburo、Suzuki Akihiro、Kobayashi Yasuhiro、Saito Makina、Seto Makoto、Ohta Takehiro
  • Journal Title: Inorganic Chemistry Volume: 60 Issue: 2 Pages: 1021-1027
  • DOI: 10.1021/acs.inorgchem.0c03123
  • Peer Reviewed
• [Journal Article] UV Resonance Raman Characterization of a Substrate Bound to Human Indoleamine 2,3-Dioxygenase 1 (2019)
  • Author(s): Sachiko Yanagisawa , Kure’e Kayama , Masayuki Hara , Hiroshi Sugimoto , Yoshitsugu Shiro , Takashi Ogura
  • Journal Title: Biophysical Journal Volume: 117 Issue: 4 Pages: 706-716
  • DOI: 10.1016/j.bpj.2019.07.017
  • Peer Reviewed
• [Journal Article] Fundamental electron-transfer and proton-coupled electron-transfer properties of Ru(iv)-oxo complexes (2019)
  • Author(s): Hiroaki Kotani, Hinatsu Shimomura, Momoka Horimoto, Tomoya Ishizuka, Yoshihito Shiota, Kazunari Yoshizawa, Sachiko Yanagisawa, Yuka Kawahara-Nakagawa, Minoru Kubo, Takahiko Kojima
  • Journal Title: Dalton Transactions Volume: 48 Issue: 35 Pages: 13154-13161
  • DOI: 10.1039/c9dt02734c
  • NAID: 120007133077
  • Peer Reviewed
• [Presentation] Stopped-flow ラマン・吸収同時測定装置の開発とそれを用 いた IDO 酵素反応追跡の試み (2021)
  • Author(s): 河村 味奈、柳澤 幸子、久保 稔
  • Organizer: 第47回生体分子科学討論会
• [Presentation] ストップトフローラマン・吸収分光計を用いたインドールアミン2,3ジオキシゲナーゼの反応中間体の研究 (2021)
  • Author(s): 河村 味奈、名定加峰、柳澤 幸子、久保 稔
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] Visible resonance Raman study on respiratory supercomplex from bovine heart mitochondria (2019)
  • Author(s): Sachiko Yanagisawa, Arisa Yamasaki, Tomomi Nakao, Satoru Shimada, Harunobu Shimomura, Takashi Oguraa and Kyoko Shinzawa-Itoh
  • Organizer: 7th Georgian Bay International Conference on Bioinorganic Chemistry
  • Int'l Joint Research / Invited