Structure and fanctional analysis of NAD^+-dependent isocitrate dehydrogenase from the chemolithotroph Aci dithiobacillus thiooxidans

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 14580648
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: OKAYAMA UNIVERSITY
• 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)
• 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)
• Project Period (FY): 2002 – 2004
• Keywords: isocitrate dehydrogenase / sulfur-oxidizing bacterium / Acidithiobatillus thiooxidans / NAD^+ / X-ray crystallographic analysis

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 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.

Research Products

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• [Journal Article] Selenophosphate genes from lung adenocarcinoma cells : Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilarion. (2004)
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  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Assay method for antitumor L-methionine γ-lyase : comprehensive kinetic analysis of the complex reaction with L-methionine (2004)
  • Author(s): Takakura, T., Akita, K., Takimoto, A., Inagaki, K., Esaki, N., Soda, K., Matsushima, K.
  • Journal Title: Anal.Biochem 327 Pages: 233-240
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Selenophoshate genes from lung adenocarcinoma cells : Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation (2004)
  • Author(s): Tamura, T., Yamamoto, S., Takahata, M., Sakaguchi, H., Tanaka, H., Inagaki, K.
  • Journal Title: Proc.Natl.Acad.Sci.USA 101 Pages: 16162-16167
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Purification and substrate characterization of α-Ketobutyrate decarboxylase from Pseudomonas putida (2003)
  • Author(s): Inoue, H., Nishito, A., Eriguchi, S., Tamura, T., Inagaki, K., Tanaka, H
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  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Recombinant expression, biochemical characterization and stabilization by proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6. (2003)
  • Author(s): Arima, J., Tamura, T., Kusakabe, H., Yagi, T., Tanaka, H., Inagaki, K
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• [Journal Article] ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein Kai C. (2003)
  • Author(s): Hayashi F., Suzuki H., Iwase R., Uzumaki T., Imada K., et al.
  • Journal Title: Genes Cells 8 Pages: 287-296
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Recombinant expression, biochemical characterization and stabilization by proteolysis of an L-glutamate oxidase from Streptomyces sp.X-119-6. (2003)
  • Author(s): Arima, J., Tamura, T., Kusakabe, H., Asiuchi, M., Yagi, T., Tanaka, H., Inagaki, K
  • Journal Title: J.Biochem. 134 Pages: 805-812
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] ATP-induced hexameric ring structure of the cyanobacterial circadian clock protein Kai C. (2003)
  • Author(s): Hayashi F., Suzuki H., Iwase R., Uzumaki T., Miyake A., Shen JR., Imada K., et al.
  • Journal Title: Genes Cells 8 Pages: 287-296
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Biochemical and molecular characterization of the NAD+-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans. (2002)
  • Author(s): Inoue, H., Tamura, T., Ehara, N., Imada, K., Tanaka, H., Inagaki, K.
  • Journal Title: FEMS Microbiol.Lett. 214 Pages: 127-132
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Biochemical and molecular characterization of the NAD+- dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans. (2002)
  • Author(s): Inoue, H., Tamura, T., Ehara, N., Nishito, A., Nakayama, Y., Maekawa, M., Imada, K., Tanaka, H., Inagaki
  • Journal Title: FEMS Microbiol.Lett. 214 Pages: 127-132
  • Description: 「研究成果報告書概要(欧文)」より