| Project/Area Number |
20370037
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Osaka University |
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Principal Investigator |
FUKUYAMA Keiichi Osaka University, 大学院・理学研究科, 教授 (80032283)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAHASHI Yasuhiro 埼玉大学, 大学院・理工学研究科, 教授 (10154874)
WADA Kei 大阪大学, 大学院・理学研究科, 助教 (80379304)
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| Co-Investigator(Renkei-kenkyūsha) |
INOMATA Katsuhiko 金沢大学, 大学院・自然科学研究科, 教授 (50110599)
SUGISHIMA Masakazu 久留米大学, 医学部, 講師 (30379292)
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| Project Period (FY) |
2008 – 2010
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| Project Status |
Completed (Fiscal Year 2010)
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| Budget Amount *help |
¥20,020,000 (Direct Cost: ¥15,400,000、Indirect Cost: ¥4,620,000)
Fiscal Year 2010: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
Fiscal Year 2009: ¥6,110,000 (Direct Cost: ¥4,700,000、Indirect Cost: ¥1,410,000)
Fiscal Year 2008: ¥7,800,000 (Direct Cost: ¥6,000,000、Indirect Cost: ¥1,800,000)
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| Keywords | ビリン還元酵素 / ビリン色素 / フェレドキシン / X線結晶解析 / クロロフィル / 光合成色素 / クロロフィルの分解 / 酵素反応機構 / PcyA / RCCR / 分子進化 |
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| Research Abstract |
In order to elucidate the molecular mechanism of PcyA, we determined the crystal structures of PcyA in complex with the reaction intermediate (18^1,18^2-dihydrobiliverdin), PcyA in complex with the synthetic analog of substrate (biliverdin XIIIα), and PcyA mutant protein of E76Q in complex with BV. These structural studies showed that the carboxyl group of Glu76 in PcyA-BV was in close proximity to the D-ring vinyl group, suggesting the presence of OH...π hydrogen bond here and that this bond is a key for the preceding reduction of the D-ring vinyl group.
When Val225 was replaced by Asp, the absorption spectrum of this mutant protein in complex with BV was very different from that of PcyA-BV. The orientation of BV in V225D-BV was inverted and the induced-fit did not occur upon BV binding. This observation presented a cautionary note about interpreting functional data derived from a mutated protein in the absence of its exact structure. In parallel we expressed, purified and crystallized cyanobacterial biliverdin reductase, an enzyme that reduces at different site of BV from PcyA, We also prepared and crystallized the Se-Met form of biliverdin reductase, and performed preliminary X-ray diffraction analyses for these crystals.
We determined the crystal structure of red chlorophyll catabolite reductase (RCCR), a key enzyme in the chlorophyll breakdown pathway. The structure was essentially the same as PcyA, implicating that RCCR belongs to the ferredoxin-dependent bilin reductase family. We also determined the crystal structures of RCCR and its mutant protein in complex with substrate, proposing the reaction mechanism as well as the active residues of RCCR.
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