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Crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase reveal mechanistic details of γ‐glutamylcysteine biosynthesis

Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) catalyzes ATP‐dependent peptide bond formation between the γ‐carboxylate of glutamate and the α‐amino group of cysteine, the first and rate‐limiting step in de novo synthesis of glutathione. Glutathione homeostasis is critically important for maintaining both intracellular redox balance and defense against oxidative or chemical stress. ScGCL was cloned, expressed, and purified to homogeneity. ScGCL crystals were obtained using the sitting‐drop vapor‐diffusion method, belong to the space group P43212, and contain a single monomer in the asymmetric unit. Structures were solved for the enzyme in the presence of glutamate and MgCl2 (2.1 Å R=18.7%, Rfree=22.5%), and also for the complex with ADP, glutamate, cysteine and MgCl2 (2.7 Å R=18.3%, Rfree=24.5%). Examination of the resulting structures provides unprecedented insight into the mechanistic details of class II glutamate cysteine ligases, and clarifies the Mg2+ dependence of the enzymatic reaction. The reported ScGCL structures were used to generate a credible homology model of human γ‐glutamate cysteine ligase. Comparison of the ScGCL structures and the human γ‐glutamate cysteine ligase model suggests that catalytic mechanism employed by ScGCL is conserved across the class II subfamily of γ‐glutamate cysteine ligases.