1997 Fiscal Year Final Research Report Summary
Hyperthermophilic enzymes suitable for functioning membranes.
| Project/Area Number |
07558128
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Biomedical engineering/Biological material science
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| Research Institution | Jichi Medical School |
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Principal Investigator |
KAGAWA Yasuo Jichi Med.Sch.Prof., 医学部, 教授 (30048962)
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| Project Period (FY) |
1995 – 1997
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| Keywords | hyperthermphile / chaperone / biomembrane / HSP70 / racemase / ATPase / reconstitution / heat shock |
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| Research Abstract |
The most important point in the application of hyperthermophilic enzymes for an artificial functional membrane is their extreme stability. The reconstitution of the artificial membrane has already been achieved, and there are many biosensors in practical uses. However, the stability of these preparations is not satisfactory.
For this reason, the research was focus on the following two hyperthermophilic enzymes :
a.The stability and functional study on the hyperthermophilic V-type ATPase, for which homological membrane ATPases have already been reported. The hyperthermophile used was Desulfurococcus which was compared with mesophiles.
b.The isolation and functional study on the hyperthermphilic chaperones which controls the higher structure of proteins.
Since both membrane ATPases and chaperones contain common ATPase structure, cloning was based on the structure. The hyperthermophiles used were Desulfurococcus and Thermococcus.
a.The identification of the stabilizing residue exchanges between the hyperthermophilic and mesophilic V-ATPases was based on the computerized homology search and the X-ray crystallography. The most abundant stabilizing residue exchange was Asp to Glu (helixstabilizing), and the next were Ser to Ala, and Gly to Ala (enhancing the internal hydrophobicity).
b.The chaperones including chaperonin and racemase was isolated after the cloning of these genes. The chaperones were quite different from HSP70 of the eubacterial thermophiles. The detailed analysis of the hyperthermophilic chaperone revealed its alpha beta subunits (J.Mol.Biol.273 : 635,1997).
Finally, these studies contributed to Nobel Prize (1997) won by J.E.Walker and P.D.Boyer on the ATPase. Walker is the coauthor of our report on X-ray crystallographic analysis of thermophilic ATPase. Boyer frequently quoted works on thermophilic ATPases in Ann. Rev.of Biochem., and will give us a lecture on this subject in Jichi Medical School in March 1998.
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[Publications] Kagawa, Y., Ohta, T., Abe, Y., Endo, H., Yohda, M., Kato, N., Endo, I., Hamamoto, T., Ichida, M., Hoaki, T.and Maruyama, T.: "Gene of heat shock protein of sulfur-dependent archaeal hyperthermophile Desulfurococcus." Biochem.Biophys.Res.Commun.214. 730-736 (1995)
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「研究成果報告書概要(欧文)」より
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[Publications] Sato, M., Ito, Y., Harada, M., Kihara, H., Tsuruta, H., Ohta, S.and Kagawa, Y.: "ATP-hydrolyzing exitation state of the reconstituted alpha 3 beta 3 complex of ATP synthase from the thermophilic bacterium PS3 : Structural characteristics shown by time-resolved small-angle X-ray scattering with synchrotron" J.Biochem.117. 113-119 (1995)
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[Publications] Jault, J-M., Matsui, T., Jault, F.M., Kaibara, C., Muneyuki, E., Yoshida, M., Kagawa, Y.and Allison, W.S.: "The alpha 3 beta 3 gamma complex of the F1-ATPase from thermophilic bacillus PS3 containing the alpha D261N substitution fails to dissociate inhibitory MgADP from a catalytic site when ATP binds to noncatalytic sites." Biochemistry. 34. 16412-16418 (1995)
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[Publications] Yohda, M., Endo, I., Abe, Y., Ohta, T., Iida, T., Maruyama, T.and Kagawa, Y.: "Gene for aspartate racemase from sulfur-dependent hyperthermophilic archaeum, Desulfurococcus Strain SY." J.Biol.Chem.271. 22017-22021 (1996)
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[Publications] Shirakihara, Y., Leslie, A.G.W., Abrahams, J.P., Walker, J.E., Ueda, T., Sekimoto, Y., Kambara, M., Saika, K., Kagawa, Y.and Yoshida, M.: "The crystal structure of the nucleotide-free alpha 3 beta 3 subcomplex of F1-ATPase from the thermophilic bacillus PS3 is a symmetric trimer." Structure. 5. 825-836 (1997)
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[Publications] Yoshida, T., Yohda, M., Iida, T., Maruyama, T.Taguchi, H., Yazaki, K., Ohta, T., Okada, M., Endo, I.and Kagawa, Y.: "Structural and functional characterization of homo-oligoneric complexes of alpha and beta chaperonin subunits from the hyperthermophilic Archaeum, Thermococcus strain KS-1." J.Molecular Biol.273. 635-645 (1997)
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