Structural Basis for the Redox Control of Plant Glutamate Cysteine Ligase

Abstract

Glutathione (GSH) plays a crucial role in plant metabolism and stress response. The rate-limiting step in the biosynthesis of GSH is catalyzed by glutamate cysteine ligase (GCL) the activity of which is tightly regulated. The regulation of plant GCLs is poorly understood. The crystal structure of substrate-bound GCL from Brassica juncea at 2.1-Å resolution reveals a plantunique regulatory mechanism based on two intramolecular redox-sensitive disulfide bonds. Reduction of one disulfide bond allows a ␤-hairpin motif to shield the active site of B. juncea GCL, thereby preventing the access of substrates. Reduction of the second disulfide bond reversibly controls dimer to monomer transition of B. juncea GCL that is associated with a significant inactivation of the enzyme. These regulatory events provide a molecular link between high GSH levels in the plant cell and associated down-regulation of its biosynthesis. Furthermore, known mutations in the Arabidopsis GCL gene affect residues in the close proximity of the active site and thus explain the decreased GSH levels in mutant plants. In particular, the mutation in rax1-1 plants causes impaired binding of cysteine.Glutathione (GSH) is the predominant non-protein thiol compound in eukaryotic and prokaryotic cells (1). Through reversible oxidation to glutathione disulfide, it acts as a major cellular redox buffer. In the plant chloroplast GSH can reach millimolar concentrations depending on diverse external stress stimuli and on the day-night transition. Light/dark-induced changes in stromal pH further modulate the redox potential of GSH (2).In plants, GSH detoxifies reactive oxygen species via the ascorbic acid-GSH-cycle (3) and glutathione peroxidases (4). It is involved in the detoxification of xenobiotics via glutathione S-transferases (5), represents the precursor for the heavy metal ions sequestering phytochelatins (6), and serves as a major defense component against a wide range of abiotic and biotic stress factors (7). GSH can post-translationally modify proteins via glutathionylation (8, 9) and may act as a signaling molecule (10, 11). Notably, GSH represents a major storage and transport form of reduced sulfur in plants (12).The biosynthesis of GSH occurs in two sequential ATP-dependent steps. Glutamate cysteine ligase (GCL, 3 EC 6.3.2.2.) produces the reaction intermediate ␥-glutamylcysteine (Scheme 1). Glutathione synthetase (EC 6.3.2.3.) then catalyzes the addition of a glycine residue.Both enzymes are encoded by single genes in Arabidopsis, belonging to the Brassicaceae family (13-15). In this plant family GCL is localized exclusively in the plastids, whereas glutathione synthetase is found both in plastids and in the cytosol (16). Under most conditions, GCL activity is rate-limiting in plant GSH biosynthesis (17). This is highlighted by significantly reduced glutathione levels in AtGCL mutants (18,19).Plant GCL protein sequences diverge from those of nonplant eukaryotic organisms (mammals, Drosophila). Although some bacteria share low sequence homology w...

Structural basis and evolution of redox regulation in plant adenosine-5′-phosphosulfate kinase

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“…1] possess cysteines that form disulfide bonds to regulate enzymatic activity in response to changes in redox environment (18,(43)(44)(45). This suggests that oxidative stresses may simultaneously affect multiple proteins across sulfur assimilation and metabolism in plants.…”

Plant homologs of the Plasmodium falciparum chloroquine-resistance transporter, Pf CRT, are required for glutathione homeostasis and stress responses

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Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes

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