Molecular design of hydrogen peroxide-dependent P450s

• Project/Area Number: 17H03087
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Nagoya University
• Principal Investigator: Watanabe Yoshihito 名古屋大学, 物質科学国際研究センター, 名誉教授 (10201245)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Project Status: Completed (Fiscal Year 2019)
• Budget Amount: ¥16,250,000 (Direct Cost: ¥12,500,000、Indirect Cost: ¥3,750,000); Fiscal Year 2019: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2018: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000); Fiscal Year 2017: ¥6,630,000 (Direct Cost: ¥5,100,000、Indirect Cost: ¥1,530,000)
• Keywords: 酵素 / シトクロムP450 / ヘム / 過酸化水素 / 水酸化反応 / カタラーゼ / 結晶構造解析 / 変異導入 / 触媒 / 鉄ポルフィリン錯体 / 酸化反応 / 不均化反応 / 酸素

Outline of Final Research Achievements

CYP152A1 (P450BSβ) and CYP152B1 (P450SPα) can generate the active species utilizing H2O2 as an oxidant and catalyze the α- or β-selective hydroxylation of long-alkyl-chain fatty acids. The substrate specificity of CYP152 family enzymes is very high. The crystal structure of P450BSβ and P450SPα indicate that the carboxylate group of palmitic acid interacting with the arginine located at the I helix (the distal side of the heme) is crucial for the generation of compound I. Owing to this unique mechanism, CYP152 family enzymes never oxidize substrate other than long-alkyl-chain fatty acid. We have demonstrated that the R242K mutant of P450BSβ and the P450SPα with corresponding mutation can catalyze non-native substrate oxidations. We have also succeeded in crystal structure analysis of the R241K mutant of P450SPα. We expect that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.

Academic Significance and Societal Importance of the Research Achievements

シトクロムP450は、高難度酸化反応が可能な有用な酸化酵素の一つですが、酸化反応を行うためには、高価な試薬であるNAD(P)Hが必要になります。安価な過酸化水素を利用することが可能なシトクロムP450もありますが、対象とする基質の選択性が高く、長鎖脂肪酸の水酸化しかできないと考えられてきました。本研究では、過酸化水素を利用可能なP450の一つのアミノ酸を違うアミノ酸に置換することで、様々な基質を水酸化できるように改変することに成功しました。

Research Products

• [Journal Article] Molecular Design and Regulation of Metalloenzyme Activities through two Novel Approaches: Ferritin and P450s (2020)
  • Author(s): Yoshihito Watanabe, Yuichiro Aiba, Shinya Ariyasu, Satoshi Abe
  • Journal Title: Bulletin of the Chemical Society of Japan Volume: 93 Issue: 3 Pages: 379-392
  • DOI: 10.1246/bcsj.20190305
  • NAID: 130007808692
  • Peer Reviewed / Open Access
• [Journal Article] Hoodwinking Cytochrome P450BM3 into Hydroxylatiing Non-Native Substrates by Exploiting Its Substrate Misrecognition (2019)
  • Author(s): Shoji,O.; Aiba, Y.; Watanabe, Y.
  • Journal Title: Acc. Chem. Res. Volume: 52(4) Issue: 4 Pages: 925-934
  • DOI: 10.1021/acs.accounts.8b00651
  • Peer Reviewed / Open Access
• [Journal Article] The effect of decoy molecules on the activity of the P450BM3 holoenzyme and a heme domain peroxygenase variant (2019)
  • Author(s): Dezvarei,S.; Shoji, O.; Watanabe, Y.; Bell, S. G.
  • Journal Title: Catalysis Communications Volume: 124 Pages: 97-102
  • DOI: 10.1016/j.catcom.2019.03.004
  • Peer Reviewed
• [Journal Article] α-Oxidative decarboxylation of fatty acids catalysed by cytochrome P450 peroxygenases yielding shorter-alkyl-chain fatty acids (2018)
  • Author(s): Hiroki Onoda, Osami Shoji, Kazuto Suzuki, Hiroshi Sugimoto, Yoshitsugu Shiro, Yoshihito Watanabe
  • Journal Title: Catalysis Science & Technology Volume: 8 Issue: 2 Pages: 434-442
  • DOI: 10.1039/c7cy02263h
  • Peer Reviewed
• [Presentation] 生体で活躍する金属イオン (2019)
  • Author(s): 渡辺芳人
  • Organizer: 立命館大学「生物無機化学フロンティア講義」
• [Presentation] 金属酵素の分子設計と機能制御 (2019)
  • Author(s): 渡辺芳人
  • Organizer: 慶應義塾大学理工学部化学科講演会
  • Invited
• [Presentation] 蛋白質を反応場とする金属触媒設計 (2019)
  • Author(s): 渡辺芳人
  • Organizer: 近畿大学「サイエンスシンポジウム 躍動する物質科学・脳科学の最前線」
  • Invited
• [Presentation] Molecular Design and Regulation of Metalloenzyme Activities (2019)
  • Author(s): 渡辺芳人
  • Organizer: 分子研シンポジウム（IMS Symposium）
  • Invited
• [Presentation] Molecular Design of Metalloenzymes (2019)
  • Author(s): Y. Watanabe
  • Organizer: KAIST 7th Kim Yong Hae Lecture
  • Int'l Joint Research / Invited
• [Presentation] 大学の現状・将来と科研費 (2019)
  • Author(s): 渡辺芳人
  • Organizer: 日本化学会 第99春季年会 (2019)
  • Invited
• [Presentation] α-Oxidative Decarboxylation Reaction of Fatty Acid Catalyzed by Fatty Acid Specific Peroxygenase (2018)
  • Author(s): H. Onoda, O. Shoji, K. Suzuki, H. Sugimoto, Y. Shiro, Y. Watanabe
  • Organizer: 9th Asian Biological Inorganic Chemistry Conference
  • Int'l Joint Research
• [Presentation] 脂肪酸特異的ペルオキシゲナーゼの反応場解析に基づいた基質寛容性の拡張 (2018)
  • Author(s): 小野田浩宜、荘司長三、杉本宏、城宜嗣、渡辺芳人
  • Organizer: 第31回生物無機化学夏季セミナー
• [Presentation] 脂肪酸特異的ペルオキシゲナーゼの基質結合状態を模した非特異的ペルオキシゲナーゼの開発 (2018)
  • Author(s): 小野田 浩宜、荘司 長三、杉本 宏、渡辺 芳人、城 宜嗣
  • Organizer: 第12回バイオ関連化学シンポジウム
• [Presentation] シトクロムP450と過酸化水素を用いた脂肪酸―炭素減炭反応機構の解析 (2018)
  • Author(s): 小野田浩宜、荘司長三、杉本宏、域宜嗣、渡辺芳人
  • Organizer: 第18回蛋白質科学会年会
• [Presentation] Molecular Design and Regulation of metalloenzymes (2017)
  • Author(s): 渡辺芳人
  • Organizer: 日本化学会 第98春季年会 (2018)
  • Invited
• [Presentation] 蛋白質反応場の活用 (2017)
  • Author(s): 渡辺芳人
  • Organizer: 日本化学会 第98春季年会 (2018)
  • Invited
• [Presentation] 化学の視点で金属酵素を設計する (2017)
  • Author(s): 渡辺芳人
  • Organizer: 第120回触媒討論会
  • Invited