2013 Fiscal Year Final Research Report

Analysis of a new substrate recognition mechanism of glutamine synthetase and its application for modulation of herbicide sensitivity

Research Project

Project/Area Number	23651219
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Living organism molecular science
Research Institution	Osaka University
Principal Investigator	HASE Toshiharu 大阪大学, たんぱく質研究所, 教授 (00127276)
Project Period (FY)	2011 – 2012
Keywords	グルタミン合成酵素 / 窒素同化 / 蛋白質工学 / 活性中心
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.