| Project/Area Number |
08455382
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
生物・生体工学
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| Research Institution | Osaka University |
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Principal Investigator |
KANAYA Shigenori Osaka University, Faculty of Engineering, Professor, 工学部, 教授 (30273585)
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| Co-Investigator(Kenkyū-buntansha) |
TAKAGI Masahiro Osaka University, Faculty of Engineering, Associate Professor, 工学部, 助教授 (00183434)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥7,000,000 (Direct Cost: ¥7,000,000)
Fiscal Year 1997: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1996: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Keywords | hyperthermophile / archaea / enzyme / stability / ion pair / gene cloning / glycerol kinase / ribonuclease HII / イオンペア-ネットワーク / ズブチリシン / 超好熱始原菌 / recA蛋白質 / アルパルチルtRNA合成酵素 / ATPase / DNase / クローニング / ポリアミン |
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| Research Abstract |
To understand the mechanism, by which the proteins from hyperthermophilic archaeon which grow at the temperature higher than 95ﾟC adapt unusually high temperatures, we have cloned the genes encoding glycerol kinase (GK) and ribonuclease HII (RNase HII) from Pyrococcus kodakaraensis KODI (Pk). these genes were overexpressed in E.coli and the recombinant enzymes were purified to homogeneity. Heat treatment was shown to be very effective to purify these enzymes, because most of the proteins from E.coli were precipitated upon heat treatment due to thermal denaturation and removed by centrifugation. Pk-GK and Pk-RNase HII are composed of 497 and 288 amino acid residues and exist in dimeric and monomeric forms, respectively. These enzymes show unusual enzyme characteristics, such as high stability against heat inactivation and broad metal ion specificity. Comparison of the amino acid compositions of these enzymes with those of the enzymes from mesophilic sources showed the contents of the charged amino acid residues in Pk-GK and Pk-RNase HII are much higher than those in E.coli GK and E.coli RNase HII,respectively. These results suggest that an increase in the number of the ion pairs or ion-pair networks contribute to increase the stabilities of Pk-GK and Pk-RNase HII,as suggested for other enzymes from hyperthermophilic archaea. Construction of a three-dimensional model for the Pk-GK structure and comparison of it with the crystal structure of E.coli GK supports this hypothesis. We are now trying to determine the crystal structures of Pk-GK and Pk-RNase HII,and to identify amino acid substitutions that makes these enzymes more stable than mesophilic ones.
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