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10个相同亚基的寡聚体结构，形成两个中心聚合环。3株突变体P146G、F150G和F150对谷氨酸的Km值比WT酶高约50倍。同时还获得了活性位点突变体H249Q，其与谷氨酸的亲和力显著降低。草甘膦(PPT)对该酶活性的抑制作用在H249Q较WT明显。PPT在P146G、F150G和Delta F150中没有观察到这种强烈的抑制作用，这表明它们对谷氨酸的低亲和力并不是由于其与活性部位的弱结合所致。活性部位和环间接触部位的双重突变导致活性的协同下降。我们发现的结构基础是基于这些突变体的晶体结构。

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.