| Project/Area Number |
11228208
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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 | Nagoya University (2002-2003)
Okazaki National Research Institutes (1999-2001) |
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Principal Investigator |
WATANABE Yoshihito Graduate School of Science, Department of Chemistry, Professor, 大学院・理学研究科, 教授 (10201245)
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| Co-Investigator(Kenkyū-buntansha) |
OZAKI Shin-ichi Graduate School of Agriculature, Yamaguchi University, Professor, 農学部, 助教授 (40280581)
OGO Seiji Graduate School of Science, Osaka University, Associate Professor, 大学院・工学研究科, 助教授 (60290904)
UENO Takafumi Research Center for material Science, Assistant Professor, 物質科学国際研究センター, 助手 (70332179)
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| Project Period (FY) |
1999 – 2002
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥61,600,000 (Direct Cost: ¥61,600,000)
Fiscal Year 2002: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2001: ¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 2000: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1999: ¥40,800,000 (Direct Cost: ¥40,800,000)
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| Keywords | myoglobin / hydroxylation / cytochrome P450 / 不斉酸化 / シッフ塩基 / 人工金属酵素 / カタラーゼ / ヘム酵素 / 活性酸素 / 金属錯体 / 酸化 / ヘム蛋白質 / ペルオキシダーゼ / 電子移動 |
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| Research Abstract |
The aromatic hydroxylation is one of the hard oxidations to proceed and we have designed a myoglobin mutant which is able to oxidize aromatic ring on the basis of the following strategies 1)Introduction of the substrate, tryptophan, at the position of phenyl alanine 43.2)Replacement of histidine 64 by asparitic acid to allow rapid reaction of the mutant with hydrogen peroxide. At the same time, Valine 68 was also replaced by isoleucine to allow hydrogen peroxide readily accessing to the active site, The oxidative modification of the protein was monitored by ESI-TOF mass measurements. Upon the addition of 1 eq. of hydrogen peroxide, 16Da increase of the molecular weight was observed. Further addition of hydrogen peroxide afforded secondary product exhibiting 32Da increase of its mass. After the digestion of the protein, a fragment including W43 was purified and characterized by MALDI TOF mass and NMR. The modified residue was W43 having 6-hydroxide. This is the first example of aromatic hydroxylation by a stoichiometric amount of hydrogen peroxide. In addition, the secondary product was characterized as 2,6-dioxo-derivative of tryptophan 43.
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