| Project/Area Number |
13125208
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | KEIO UNIVERSITY |
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Principal Investigator |
SHIMADA Hideo Keio University, School of Medicine, Associate Professor, 医学部, 助教授 (80095611)
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| Co-Investigator(Kenkyū-buntansha) |
FUJII Hiroshi Okazaki National Research Institutes, Institute for Molecular Science and Center for Integrative Bioscience, Associate Professor, 統合バイオサイエンスセンター, 助教授 (80228957)
HAYASHI Takashi Kyushu University, Graduate School of Engineering, Associate Professor, 大学院・工学研究院, 助教授 (20222226)
MUKAI Kuniaki Keio University, School of Medicine, Instructor, 医学部, 助手 (80229913)
菱木 貴子 慶應義塾大学, 医学部, 助手 (10338022)
江川 毅 慶應義塾大学, 医学部, 助手 (10232935)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥40,600,000 (Direct Cost: ¥40,600,000)
Fiscal Year 2003: ¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 2002: ¥8,300,000 (Direct Cost: ¥8,300,000)
Fiscal Year 2001: ¥24,700,000 (Direct Cost: ¥24,700,000)
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| Keywords | composite biocatalyst / gene therapy / heme / myoglobin / cytochrome oxidase / proton pump / membrane protein / cell-free protein synthesis / 膜たんぱく質複合体 / ポルフィセン / 亜硝酸還元酵素 / ペルオキシダーゼ / 膜タンパク質複合体 / 部位特異的アミノ酸置換 / ミトコンドリア / シトクロムP450 / オーダーメイド / 水素イオン供給 / 過酸化水素 / ペルオキシゲナーゼ / 亜硝酸イオン還元酵素 |
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| Research Abstract |
As a research intended to construct heme-based highly efficient biocatalyst, we have developed a functional expression system of bovine/human hybrid cytochrome oxidase in human HeLa cells by integrating the bovine mitochondrial DNA derived subunit I gene into the cell genome, whose product is the largest with 12 trans membrane helices in the 13 different enzyme-subunits and essential to the function. The expression and subsequent import of subunit I into the inner membrane of mitochondria permit the association with the other 12 human subunits leading to the hybrid enzyme having the same dioxygen reduction and proton pumping activities as those of the human enzyme, In this system, the hybrid dominates over the human enzyme. By mutating the subunit I, we have found the key residue for the proton pumping and also demonstrated that the proton pumping can be uncoupled from the dioxygen reduction. The methods we have developed ale applicable to cure mitochondrial diseases caused by mal-func
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tion of mitochondrial DNA derived membrane proteins. We have also developed the cell-free functional synthesis of Paracoccus denitrificans cytochrome oxidase by exogenously adding the prosthetic group into E. coli reaction system.
The prosthetic group, protoheme, of myoglobin (Mb) can be chemically modified to functionalize the protein as monooxygenase, peroxygenase and peroxidase. Incorporation of unnatural heme, metalloporphycene, into Mb turned out to enhance the oxygen affinity to 1000-fold. Protein engineering of Mb with the above porphycene powers up the oxidizing activity enormously to allow the oxidative destruction of bisphenol A, a notable endocrine disrupter. These results revealed that unnatural heme including chemically modified are powerful to develop a protein-based efficient catalyst.
Our in vivo and in vitro functional expression system of cytochrome oxidase pave the way to produce a highly efficient biocatalyst from highly hydrophobic membrane proteins by combining the protein engineering with an incorporation of unnatural heme into the active center. Less
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