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

• Antragsteller: Professor Dr. Thomas Rausch; Professor Dr. Klaus Scheffzek
• Fachliche Zuordnung: Biochemie und Biophysik der Pflanzen
• Förderung: Förderung von 2005 bis 2009
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 15479759

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.

• DFG-Verfahren: Sachbeihilfen