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

植物谷氨酰胺合成酶（GS）具有低聚结构，具有10个相同的亚基，形成两个五聚体环。我们在玉米细胞质GS （GS1a）的环间接触位点进行了几个氨基酸替换。3个突变体P146G、F150G和F150表现出较低的谷氨酸活性，其谷氨酸Km值比WT酶高约50倍。一个活性位点突变体H249Q对谷氨酸的亲和力显著降低。在H249Q中，phosphinothricin （PPT）对酶活性的抑制作用比在WT中更为显著。PPT对P146G、F150G和delta F150的抑制作用不明显，说明它们对谷氨酸的低亲和力不是由于与活性位点的弱结合所致。活性位点和环间接触位点的双重突变导致了活性的协同下降。基于这些突变体的晶体结构提出了我们发现的结构基础。

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.