Analysis of a new substrate recognition mechanism of glutamine synthetase and its application for modulation of herbicide sensitivity
| Project/Area Number |
23651219
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Living organism molecular science
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| Research Institution | Osaka University |
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Principal Investigator |
HASE Toshiharu 大阪大学, たんぱく質研究所, 教授 (00127276)
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| Project Period (FY) |
2011 – 2012
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| Project Status |
Completed (Fiscal Year 2013)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2011: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | グルタミン合成酵素 / 窒素同化 / 蛋白質工学 / 活性中心 |
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| Research Abstract |
Plant glutamine synthetase (GS) has an oligomeric structure with 10 identical subunits to form two pentermer rings.We made several amino acid substitutions at the inter-ring contact site of maize cytosolic GS (GS1a). Three such mutants, P146G, F150G and F150 showed a low activity and their Km value for glutamate became higher by ca 50 fold than that of WT enzyme. An active site mutant, H249Q was also made, whose affinity for glutamate was remarkably decreased. Inhibitory effect of phosphinothricin (PPT) on the enzyme activity was more remarkable in H249Q than in WT. Such strong inhibition by PPT was not observed in P146G, F150G or delta F150, suggesting that their low affinity for glutamate was not due to its weak binding to the active site. Double mutation at the active site and at the inter-ring contact site resulted in a synergistic drop in the activity. The structural basis for our findings is proposed based on crystal structures of these mutants.
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Report
(4 results)
Research Products
(6 results)
