| Project/Area Number |
16570189
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Evolutionary biology
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| Research Institution | Tokyo University of Pharmacy and Life Sciences |
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Principal Investigator |
YAMAGISHI Akihiko Tokyo Univ.Pharm.Life Sci, Sch.Life Sci., Prof., 生命科学部, 教授 (50158086)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | common ancestor / hyperthermophile / Archaea / Eubacteria / Thermostability of enzyme / isopropylmalate dehydrogenase / 超好熱菌 / 全生物の共通の祖先 / 祖先型変異酵素 / イソクエン酸脱水素酵素 / グリシルtRNA合成酵素 |
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| Research Abstract |
In order to test experimentally the hypothesis that the common ancestor of all the living organisms on the earth was hyperthermophilic, we designed and conducted a series of experiments. We constructed the phylogenetic tree of the two closely related enzymes : isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase. The ancestral sequence of these enzymes possessed by the common ancestor of all the living organisms was inferred using the phylogenetic tree. Each ancestral amino acid residue was individually introduced to the IPMDH of an extreme thermophile Thermus thermophilus as a mutation. The mutant IPMDHs was expressed in Escherichia coli and purified. The thermostability of each ancestral mutant IPMDH was estimated. The 6 out of 12 ancestral mutants showed hither thermal stability than the original T.thermophilus IPMDH. However, there still remains the possibility that the effect of ancestral mutation resulted from the fact that the ancestral residue is conserved. However, the statistic analysis showed that the stabilization effect can be attributed to the antiquity of the residues but not to the conservation of the residues.
The multiple ancestral mutant IPMDHs each having multiple ancestral mutant residues were constructed and analyzed. The multiple ancestral mutant IPMDHs showed higher stability than single point ancestral mutant IPMDHs. The multiple and single-point ancestral mutant IPMDHs have simillar or even improved enzymatic activity.
The ancestral NDT having ancestral residues were constructed and showed high thermal stability.
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