Development of novel radical enzymes bearing unusual protein-derived cofactor

• Project/Area Number: 15K13743
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Bio-related chemistry
• Research Institution: Osaka University
• Principal Investigator: FUJIEDA Nobutaka 大阪大学, 工学研究科, 助教 (00452318)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2015: ¥2,730,000 (Direct Cost: ¥2,100,000、Indirect Cost: ¥630,000)
• Keywords: 人工金属酵素 / 翻訳後化学修飾 / ラジカル酵素

Outline of Final Research Achievements

Galactose oxidase and copper amine oxidase have the protein-derived redox-active organic cofactors generated by post-translational modification near the catalytic active center. There are several lines of evidences that the copper ion plays an important role in the formation of these cofactors. The copper-substituted variants of cupin proteins have been prepared by using an E. coli expression system with a copper-containing medium. Furthermore, we introduced Tyr and/or Cys residues into several site surrounding copper center and confirmed the formation of Tyr-Cys by spectroscopic method as well as X-ray crystal structural analysis.

Research Products

• [Journal Article] Controlling dicopper protein functions (2016)
  • Author(s): Nobutaka Fujieda and Shinobu Itoh
  • Journal Title: Bull. Chem. Soc. Jpn. Volume: 89 Issue: 7 Pages: 733-742
  • DOI: 10.1246/bcsj.20150444
  • NAID: 130005253042
  • Peer Reviewed
• [Journal Article] Enzyme Repurposing of a Hydrolase as an Emergent Peroxidase upon Metal Binding (2015)
  • Author(s): Nobutaka FUJIEDA, Jonas SCHATTI, Edward STUTTFELD, Kei OHKUBO, Timm MAIER, Shunichi FUKUZUMI, and *Thomas R. WARD
  • Journal Title: Chem. Sci Volume: 6 Issue: 7 Pages: 4060-4065
  • DOI: 10.1039/c5sc01065a
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Tyr-Cys架橋を有する銅含有タンパク質の調製と酸化活性 (2017)
  • Author(s): 山脇沙耶香・谷口勇希・藤枝伸宇・伊東 忍
  • Organizer: 日本化学会第97回春季年会
  • Place of Presentation: 慶應義塾大学日吉キャンパス
  • Year and Date: 2017-03-16
• [Presentation] 人工金属酵素活性中心における翻訳後Tyr-Cys結合形成とその機能解明 (2016)
  • Author(s): 藤枝伸宇・谷口勇希・山脇沙耶香・伊東 忍
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 石川県立音楽堂
  • Year and Date: 2016-09-07
• [Presentation] 活性部位にチロシン－システイン架橋構造を有する人工金属タンパク質の調製と性質 (2016)
  • Author(s): 山脇沙耶香・谷口勇希・藤枝伸宇・伊東 忍
  • Organizer: 第10回バイオ関連化学シンポジウム
  • Place of Presentation: 石川県立音楽堂
  • Year and Date: 2016-09-07
• [Presentation] 配位子としてのタンパク質：機能性アミノ酸架橋の創製 (2016)
  • Author(s): 藤枝伸宇
  • Organizer: ミニワークショップ「生体機能関連化学の最先端」
  • Place of Presentation: 東京工業大学すずかけ台キャンパス
  • Year and Date: 2016-06-25
  • Invited
• [Presentation] Creation of Artificial Metalloprotein Bearing the Tyr-Cys Covalent Linkage (2016)
  • Author(s): 藤枝伸宇・谷口勇希・山脇沙耶香・伊東 忍
  • Organizer: 第26回金属の関与する生体関連反応シンポジウム
  • Place of Presentation: 北海道大学
  • Year and Date: 2016-06-17
• [Presentation] Development of an Artificial Metalloenzyme Bearing Datively Anchored Osmium Catalytic Center (2016)
  • Author(s): Nobutaka FUJIEDA, Takumi NAKANO, Haruna ICHIHASHI, Hideki SUGIMOTO, and Shinobu ITOH
  • Organizer: 96th Annual Meeting of Chemical Society of Japan
  • Place of Presentation: 同志社大学 京田辺キャンパス（京都府京田辺市）
  • Year and Date: 2016-03-24
• [Presentation] Metalloenzyme Using Cupin Superfaimly Protein as Metal-binding Platform (2015)
  • Author(s): Nobutaka FUJIEDA, Takumi NAKANO, Haruna ICHIHASHI, Yuma MORIMOTO, Hideki SUGIMOTO, and Shinobu ITOH
  • Organizer: 17th International Biological Inorganic Chemistry Conference
  • Place of Presentation: China National Convvention Center (Beijin, China)
  • Year and Date: 2015-07-20
  • Int'l Joint Research
• [Presentation] Semisynthetic Artificial Metalloenzyme Using Cupin Superfaimly Protein as Metal-binding Platform (2015)
  • Author(s): Nobutaka FUJIEDA, Takumi NAKANO, Haruna ICHIHASHI, Yuki TANIGUCHI, Hideki SUGIMOTO, and Shinobu ITOH
  • Organizer: 第25回金属の関与する生体関連反応シンポジウム
  • Place of Presentation: 長崎大学 文教地区キャンパス（長崎県長崎市）
  • Year and Date: 2015-05-30
• [Presentation] 配位子としてのタンパク質―人工金属酵素創製に向けて― (2015)
  • Author(s): 藤枝伸宇
  • Organizer: 第52回錯体化学若手の会・近畿支部勉強会
  • Place of Presentation: 大阪大学吹田キャンパス（大阪府吹田市）
  • Year and Date: 2015-05-16
  • Invited
• [Book] Fungal Pro-tyrosinase, Handbook of Metalloproteins to the Encyclopedia of Inorganic and Bioinorganic Chemistry (2015)
  • Author(s): Fujieda, N. and Itoh, S.
  • Total Pages: 8
  • Publisher: John Wiley & Sons