Solving the 3D-structure of plant-type y-glutamylcysteine sythetase (GSH1): Structural basis for post-translational redox control and evolutionary relationship with proteobacterial GSH1 proteins

GSH1 （y-谷氨酰半胱氨酸合成酶，EC 6.3.2.2.）是谷胱甘肽（GSH）生物合成的关键酶。了解其复杂的翻译后调控对于通过转基因方法优化作物中基于谷胱甘肽的胁迫防御至关重要。植物的GSH1蛋白序列与动物和真菌的GSH1蛋白序列有很大的不同，但与某些蛋白质细菌的GSH1酶的序列相似性超过50%。在离体条件下，植物GSH1酶可以形成分子内二硫桥，gsh10x的活性要高10倍。然而，这种氧化还原敏感性与GSH1体内调节的相关性尚未得到证实。我们将从芥菜和油菜黄单胞菌和农杆菌两种变形菌中（60%和57%序列与BJGSH1同源）解析GSH1蛋白的晶体结构，并解析其结构。结晶将在没有/存在底物和/或抑制剂（谷氨酸、半胱氨酸、MgATP、BSO）的情况下进行。保存在植物GSH1酶中的胱氨酸残基，包括那些也存在于蛋白质细菌GSH1蛋白中的胱氨酸残基，将发生突变，并将确定对蛋白质结构和酶活性的影响。由于植物GSH1酶局限于质体，因此将分析塑性氧化还原调节剂（GSH，硫氧还蛋白）在体外氧化/还原重组GSH1的潜力。为了确定不同生长条件下GSH1在体内的氧化还原状态（光/暗过渡，应激暴露），将开发一种提取方案来分离GSH10x和GSH1red。

GSH1 (y-glutamylcysteine synthetase, EC 6.3.2.2.) is the key enzyme for glutathione (GSH) biosynthesis. Understanding its complex post-translation regulation is pivotal to the optimization of GSH-based stress defence in crop plants via transgenic approaches. Plant GSH1 protein sequences differ strongly from their animal and fungal counterparts, but share more than 50% sequence identity with certain proteobacterial GSH1 enzymes. In vitro, plant GSH1 enzymes may form intramolecular disulfide-bridge(s), GSH1OX being 10-fold more active. However, the relevance of this redox-sensitivity for the in vivo regulation of GSH1 has not been demonstrated. We will solve the crystal structures of GSH1 proteins from Brassica juncea and from two proteobacteria, Xanthomonas campestris and Agrobacterium tumefaciens (60% and 57% sequence identity with BJGSH1) and resolve their structures. Crystallization will be performed in the absence/presence of substrates and/or inhibitors (glutamate, cysteine, MgATP, BSO). Cys residues conserved in plant GSH1 enzymes, including those present also in the proteobacterial GSH1 proteins, will be mutated, and the impact on protein structure and enzyme activity will be determined. As plant GSH1 enzymes are confined to plastids, plastidic redox regulators (GSH, thioredoxin) will be analyzed for their potential to oxidize/reduce recombinant GSH1 in vitro. To determine the redox state of GSH1 in vivo under different growth conditions (light/dark transition, stress exposure), an extraction protocol will be developed to separate GSH10x from GSH1red.