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

谷氨酰胺合成酶新底物识别机制分析及其在除草剂敏感性调节中的应用

• 批准号: 23651219
• 负责人: HASE Toshiharu
• 金额: $ 2.5万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Challenging Exploratory Research
• 财政年份: 2011
• 资助国家: 日本
• 起止时间: 2011 至 2012
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-23651219/
• 关键词: グルタミン合成酵素; 窒素同化; 蛋白質工学; 活性中心

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.

植物谷氨酰胺合成酶（GS）具有一个寡聚结构，具有10个相同的亚基，形成两个pentermer环。我们在玉米胞质GS（GS1A）的玉米间接触部位进行了几个氨基酸取代。三个这样的突变体P146G，F150G和F150的活性较低，谷氨酸的KM值比WT酶高出Ca 50倍。还制造了一个活跃的位点突变体H249Q，其对谷氨酸的亲和力明显降低。磷酸蛋白（PPT）对酶活性的抑制作用在H249Q中比在WT中更为明显。在P146G，F150G或DELTA F150中未观察到PPT的这种强抑制作用，这表明它们对谷氨酸的亲和力低是由于其与活性位点的弱结合所致。活跃部位和环间接触部位的双重突变导致活动的协同下降。根据这些突变体的晶体结构提出了我们发现的结构基础。

项目成果

Site-directed mutation of plant glutamine synthetase resulting in differential effect on the recognition of glutamate and inhibition by substrate analogues

植物谷氨酰胺合成酶的定点突变导致对谷氨酸的识别和底物类似物的抑制产生不同的影响

• 发表时间: 2013
• 作者: T. Ozaki;M. Kusunoki;A. Nakagawa;T. Hase
• 通讯作者: T. Hase

Study of substrate recognition mechanism of plant glutamine synthetase : Identification of the region distant from the active site involved in high affinity of glutamate

植物谷氨酰胺合成酶底物识别机制研究：远离谷氨酸高亲和力活性位点的区域的鉴定

• 发表时间: 2011
• 作者: T. Ozaki;A. Nakagawa;T. Hase;M. Kusunoki
• 通讯作者: M. Kusunoki

Distant residues of maize glutamine synthetase affecting substrate

影响底物的玉米谷氨酰胺合成酶的远处残基

• 发表时间: 2011
• 作者: Masami Kusunoki;Takeshi Ozaki, Atsushi Nakagawa, Toshiharu Hase
• 通讯作者: Takeshi Ozaki, Atsushi Nakagawa, Toshiharu Hase

Arabidopsis thaliana ASN2 encoding asparagine synthetase is involved in the control of nitrogen assimilation and export during vegetative growth

• DOI: 10.1111/j.1365-3040.2012.02576.x
• 发表时间: 2013-02-01
• 期刊: PLANT CELL AND ENVIRONMENT
• 影响因子: 7.3
• 作者: Gaufichon, Laure;Masclaux-Daubresse, Celine;Suzuki, Akira
• 通讯作者: Suzuki, Akira

トウモロコシ由来グルタミン合成酵素 GS1aと GS1d 活性の変化の構造学的研究

玉米源谷氨酰胺合成酶 GS1a 和 GS1d 活性变化的结构研究

• 发表时间: 2011
• 作者: 尾崎健;中川敦史;長谷俊治;小林大千;楠木正巳
• 通讯作者: 楠木正巳