Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana
Thiol-based regulation of redox-active glutamate-cysteine ligase from Arabidopsis thaliana
复制标题
DOI：
10.1105/tpc.107.052597
复制
发表时间：
2007-08-01
期刊：
影响因子：
11.6
通讯作者：
Jez, Joseph M.
中科院分区：
文献类型：
--
作者：
Hicks, Leslie M.;Cahoon, Rebecca E.;Jez, Joseph M.
谷胱甘肽的生物合成是植物胁迫反应网络中的一个关键组成部分，它可以抵消氧化损伤，维持细胞内的氧化还原环境。利用质谱学和定点突变相结合的方法，研究了拟南芥谷氨酸-半胱氨酸连接酶(GCL)对氧化还原环境变化的响应。质谱仪鉴定了GCL中的两个二硫键(Cys186-Cys406和Cys349-Cys364)。Cys-349或Cys-364突变为Ser使反应速度降低两倍，但Ser替代Cys-186或Cys-406使活性降低20倍，并取消对氧化还原环境变化的反应。氧化还原滴定表明，调节二硫键具有与其他已知的氧化还原响应植物蛋白相当的中点电位。Cys-102、Cys-251、Cys-349或Cys-364的突变不改变对氧化还原环境的反应，表明活性的调节依赖于Cys186-Cys406二硫键。体内GCL的分析表明，多重氧化胁迫改变了氧化(活性)和还原(非活性)酶的分布，并且这种变化与GCL活性的增加有关。基于硫醇的GCL调控提供了一种翻译后的机制来调节酶的活性，以响应体内的氧化还原环境，并表明氧化信号在维持植物谷胱甘肽的动态平衡中发挥了作用。
Glutathione biosynthesis is a key component in the network of plant stress responses that counteract oxidative damage and maintain intracellular redox environment. Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of Arabidopsis thaliana glutamate-cysteine ligase (GCL) to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186- Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys-349 or Cys-364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys-186 or Cys-406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Mutation of Cys-102, Cys-251, Cys-349, or Cys-364 did not alter the response to redox environment, indicating that modulation of activity depends on the Cys186- Cys406 disulfide bond. In vivo analysis of GCL in Arabidopsis root extracts revealed that multiple oxidative stresses altered the distribution of oxidized ( active) and reduced ( inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants.