| Project/Area Number |
14580648
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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 |
Functional biochemistry
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
INAGAKI Kenji Okayama University, The Graduate School of Natural Science and Technology, Professor, 大学院・自然科学研究科, 教授 (80184711)
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| Co-Investigator(Kenkyū-buntansha) |
TAMURA Takashi Okayama University, Associate Professor, 助教授 (40253009)
IMADA Katsumi Osaka University, Associate Professor, 大学院・生命機能研究科, 助教授 (40346143)
TANAKA Hidehiko Okayama University, Professor Emeritus, 名誉教授 (90065912)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | isocitrate dehydrogenase / sulfur-oxidizing bacterium / Acidithiobatillus thiooxidans / NAD^+ / X-ray crystallographic analysis / イソクエン酸脱水素酵素 / 基質・補酵素認識 |
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| Research Abstract |
1)Functional analysis of Isocitrate Dehydrogenase from Acidithiobacillus thiooxidans
Isocitrate dehydrogenase(ICDH) catalyzes the oxidative decarboxylation of D-isocitrate to 2-oxoglutarate and CO_2 with NAD^+ or NADP^+ as cofactor in the TCA cycle. ICDH from Acidithiobacillus thiooxidans is NAD^+-dependent, although most bacteria contain NADP^+-dependent ICDH. ICDH and 3-isopropylmalate dehydrogenase(IPMDH) which generally shows NAD^+-dependency belong to a class of protein family, decarboxylating dehydrogenases, that lack a typical βαβ nucleotide-binding fold which is commonly present in most dehydrogenases. Although these enzymes show high sequence homology and similarity in their 3-D structure, amino acid sequence of substrate-binding site is different. These enzymes also provide an attractive model system to study the coenzyme and substrate recognition. So we tried to build a product system which produces amount of enzyme (ICDH) by use of E.coli JM109 transformed with pkk-ICDH. And
… More
we purified ICDH by centrifugation, heat-shock, DEAE-Toyopearl and Sephacryl S-200HR. As a result of purification we obtained purified enzyme by 57.8-fold. We also analyzed the enzyme with thermostability, pH stability etc of At-ICDH. In result, ICDH from Acidithiobacillus thiooxidans is superior to that of ICDH from yeast.
2)Crystallography and Quantum Enzyme Chemistry of At-ICDH
Bacterial ICDHs normally require NADP as the cofactor, but At-ICDH is specific to NAD. We obtained bi-pyramidal crystals of ICDH-NAD complex with a space group P43212 and the unit cell of a=b=125.99Å, c=268.35Å. The structure was solved by SAD method using Os-derivative crystals and refined to 1.9Å resolution. Although most of enzyme-bound nicotinamide cofactors found in PDB have the amide NH2 group in trans to the C4 carbon of nicotinamide ring, we determined in At-ICDH the nitrogen was in cis to C4 carbon slanting by 23° toward substrate. Electro-potentials of hydrogen atoms on nicotinamide ring were computed along with the amide rotation using MNDO Hamiltonian. A hydrogen atom on C4 atom had the greatest negative charge when amide NH2 group was in the cis conformation with dihedral angle of 23°. Negative charge induced by the amide NH2 appeared to suggest a catalytic significance in stabilizing the transition state during the hydride transfer catalysis. Less
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