Institut de Recherche en Biologie Végétale, Université de Montréal, Montréal, Québec, Canada.
Morden Research and Development Centre, Agriculture and Agri-Food Canada, Morden, Manitoba, Canada.
PLoS One. 2018 Sep 25;13(9):e0204530. doi: 10.1371/journal.pone.0204530. eCollection 2018.
In plant cells, many stresses, including low oxygen availability, result in a higher production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). These molecules can lead to redox-dependent post-translational modification of proteins Cys residues. Here, we studied the effect of different redox modifications on alcohol dehydrogenase (ADH) from Arabidopsis thaliana. ADH catalyzes the last step of the ethanol fermentation pathway used by plants to cope with energy deficiency during hypoxic stress. Arabidopsis suspension cell cultures showed decreased ADH activity upon exposure to H2O2, but not to the thiol oxidizing agent diamide. We purified recombinant ADH and observed a significant decrease in the enzyme activity by treatments with H2O2 and diethylamine NONOate (DEA/NO). Treatments leading to the formation of a disulfide bond between ADH and glutathione (protein S-glutathionylation) had no negative effect on the enzyme activity. LC-MS/MS analysis showed that Cys47 and Cys243 could make a stable disulfide bond with glutathione, suggesting redox sensitivity of these residues. Mutation of ADH Cys47 to Ser caused an almost complete loss of the enzyme activity while the Cys243 to Ser mutant had increased specific activity. Incubation of ADH with NAD+ or NADH prevented inhibition of the enzyme by H2O2 or DEA/NO. These results suggest that binding of ADH with its cofactors may limit availability of Cys residues to redox modifications. Our study demonstrates that ADH from A. thaliana is subject to different redox modifications. Implications of ADH sensitivity to ROS and RNS during hypoxic stress conditions are discussed.
在植物细胞中，许多应激条件，包括低氧可用性，会导致活性氧（ROS）和活性氮（RNS）的产生增加。这些分子可以导致蛋白质半胱氨酸残基的氧化还原依赖性翻译后修饰。在这里，我们研究了不同氧化还原修饰对拟南芥醇脱氢酶（ADH）的影响。ADH 催化植物利用乙醇发酵途径的最后一步，以应对缺氧应激期间的能量缺乏。拟南芥悬浮细胞培养物在暴露于 H2O2 时显示 ADH 活性降低，但对硫醇氧化剂二酰胺（DEANA/NO）没有影响。我们纯化了重组 ADH，并观察到 H2O2 和二乙胺 NONOate（DEA/NO）处理显著降低了酶活性。导致 ADH 与谷胱甘肽之间形成二硫键的处理（蛋白质 S-谷胱甘肽化）对酶活性没有负面影响。LC-MS/MS 分析表明，Cys47 和 Cys243 可以与谷胱甘肽形成稳定的二硫键，表明这些残基的氧化还原敏感性。ADH Cys47 突变为丝氨酸几乎完全丧失了酶活性，而 Cys243 突变为丝氨酸的突变体具有增加的比活性。将 ADH 与 NAD+或 NADH 孵育可防止 H2O2 或 DEA/NO 抑制酶活性。这些结果表明，ADH 与其辅助因子的结合可能限制了 Cys 残基对氧化还原修饰的可用性。我们的研究表明，拟南芥的 ADH 受到不同的氧化还原修饰。讨论了 ADH 在缺氧应激条件下对 ROS 和 RNS 的敏感性的影响。
