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

GSH 1（γ-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是通过转基因方法优化作物中基于GSH的胁迫防御的关键。植物GSH 1蛋白序列与动物和真菌的对应物有很大的不同，但与某些变形菌GSH 1酶有超过50%的序列同一性。在体外，植物GSH 1酶可形成分子内二硫键桥，GSH 1 OX活性高10倍。然而，这种氧化还原敏感性的GSH 1的体内调节的相关性尚未得到证明。我们将解决GSH 1蛋白的晶体结构从芥菜和两个变形菌，野油菜黄单胞菌和根癌农杆菌（60%和57%的序列同一性与BJGSH 1），并解决他们的结构。将在不存在/存在底物和/或抑制剂（谷氨酸盐、半胱氨酸、MgATP、BSO）的情况下进行结晶。将突变植物GSH 1酶中保守的Cys残基，包括也存在于变形菌GSH 1蛋白中的Cys残基，并确定对蛋白质结构和酶活性的影响。由于植物GSH 1酶仅限于质体，质体氧化还原调节剂（GSH，硫氧还蛋白）将被分析其在体外氧化/还原重组GSH 1的潜力。为了确定GSH 1在不同生长条件（光/暗转换、应力暴露）下的体内氧化还原状态，将开发一种提取方案以分离GSH 10 x和GSH 1 red。

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.