2000 Fiscal Year Final Research Report Summary
ANALYSIS OF SUBSTRATE RECOGNITION MECHANISM OF ISOPROPYLMALATE DEHYDROGENASE
| Project/Area Number |
10680612
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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 |
TANAKA Hidehiko FACULTY OF AGRICULTURE, OKAYAMA UNIVERSITY, PROFESSOR, 大学院・自然科学研究科, 教授 (90065912)
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| Co-Investigator(Kenkyū-buntansha) |
TAMURA Takashi FACULTY OF AGRICULUTURE, OKAYAMA UNIVERSITY, ASSOCIATE PROFESSOR, 農学部, 助教授 (40253009)
INAGAKI Kenji FACULTY OF AGRICULTURE, OKAYAMA UNIVERSITY, PROFESSOR, 農学部, 教授 (80184711)
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| Project Period (FY) |
1998 – 2000
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| Keywords | ISOPROPYLMALATE DEHYDROGENASE / ISOCITRATE DEHYDROGENASE / SUBSTRATE RECOGNITION MECHANISM / COENZYME SPECIFICITY / X-RAY CRYSTALLOGRAPHY |
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| Research Abstract |
1) Structure of 3-isopropylmalate dehydrogenase from Thiobacillus ferrooxidans at 2.0 Åresolution.
3-Isopropylmalate dehydrogenase (IPMDH) gene was cloned and sequenced from Thiobacillus ferrooxidans (Tf). We have determined the crystal structures of Tf-IPMDH and the substrate binding mechanism have been solved. IPMDH shows a marked similarity to isocitrate dehydrogenase (ICDH) in its structural framework and catalytic mechanism, and is classified into a unique group of decarboxylating dehydrogenases. The difference between their substrates is the γ moiety attached to 2R-malate.
The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the γ-isopropyl group. The γgroup is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92. The Cβ and Cγ atoms of Glu88 interact with the γ-isopropyl moiety of 3-isopropylmalate (IPM) and are central to the recognition of substrate.
2) Mutagenesis of
… More
substrate recognition site of Tf-IPMDH.
We have changed γmoiety recognition site of Tf-IPMDH (Ala72〜Leu92) to that of Ec-ICDH (Pro102〜Val116). We analyzed the substrate specificity of the mutant enzyme. We have been investigated whether the higher-order structure for the mutant enzyme is stabilized because about ten amino acid residues of leuB gene are substituted. In result, The mutant enzyme's loop regions will be distorted slightly, but we expected the structure of the mutant enzyme was stabilized and began on this theme. The mutant enzyme was expressed as soluble protein in E.coli.
3 )Structure of NAD dependent ICDH from Thiobacillus thiooxidans and coenzyme recognition mechanism.
NAD dependent ICDH gene from Thiobacillus thiooxidans (Tt) was cloned and sequenced and the coenzyme recognition mechanism was analyzed. The amino acid sequence had a high degree of similarity to the NADP dependent ICDH and IPMDH.Tt-ICDH is 59.2%, and 22.6% identical to Ec-ICDH and Tf-IPMDH, respectively. It is expected that the tertiary structure of Tt-ICDH is similar to Ec-ICDH and Tf-IPMDH, because the regions composed a deduced secondary structure (α-helix and β-sheet) of Tt-ICDH are much alike.
Tt-ICDH has conserved Asp357 which is significant of the NAD recognition in the coenzyme binding site alignment. On the other hand, Tt-ICDH lacked for a tyrosine residue which was conserved in NADP-ICDH and recognized phosphate of NADP+. Thus, it is revealed that this enzyme has similar coenzyme binding site to that of the IPMDH's. Less
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[Publications] Motoshima,M.,K.Inagaki,T.Kumasaka,M.Furuichi,H.Inoue,T.Tamura,N.Esaki,K.Soda,N.Tanaka,M.Yamamoto,and H.Tanaka: "Crystal Structure of the Pyridoxal-5′-phosphate Dependent L-methionine γ-lyase from Pseudomonas putida"J.Biochem.,. 128・3. 349-354 (2000)
Description
「研究成果報告書概要(和文)」より
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[Publications] Motoshima, M., Kenji Inagaki, T.Kumasaka, M.Furuichi, Hiroyuki Inoue, Takashi Tamura, Nobuyoshi Esaki, Kenji Soda, N.Tanaka, M.Yamamoto, and Hidehiko Tanaka: "Crystal Structure of the Pyridoxal-5'-phosphate Dependent L-methionine γ-lyase from Pseudomonas putida."J.Biochem.. 128. 349-354 (2000)
Description
「研究成果報告書概要(欧文)」より
