Mechanistic Details of Glutathione Biosynthesis Revealed by Crystal Structures of Saccharomyces cerevisiae Glutamate Cysteine Ligase

Abstract

Glutathione is a thiol-disulfide exchange peptide critical for buffering oxidative or chemical stress, and an essential cofactor in several biosynthesis and detoxification pathways. The ratelimiting step in its de novo biosynthesis is catalyzed by glutamate cysteine ligase, a broadly expressed enzyme for which limited structural information is available in higher eukaryotic species. Structural data are critical to the understanding of clinical glutathione deficiency, as well as rational design of enzyme modulators that could impact human disease progression. Examination of the hGCLC model suggests that post-translational modifications of cysteine residues may be involved in the regulation of enzymatic activity, and elucidates the molecular basis of glutathione deficiency associated with patient hGCLC mutations.Glutathione, ␥-glutamylcysteinyl glycine, is a low molecular weight thiol, central to maintenance of redox homeostasis. Among its normal functions are the scavenging of reactive oxygen and nitrogen species (1), storage and transport of cysteine (2, 3), leukotriene, and prostaglandin biosynthesis (4, 5), and regulation of enzyme activity via reduction of disulfide bonds and glutathionylation (6, 7). Disruption of glutathione metabolism is associated with the progression of AIDS, cancer, and neurodegenerative conditions such as Parkinson and Alzheimer disease (8 -12). Polymorphisms that reduce activity of glutamate cysteine ligase (GCL), 2 the first and rate-limiting enzyme in de novo synthesis of glutathione, are correlated with reduced glutathione levels in patients with hemolytic anemia, schizophrenia and other neurological disorders (13-16). Given its importance both in normal and disease states, there is considerable interest in the development of novel compounds that could be used to modulate intracellular glutathione levels, potentially via GCL.Glutathione is synthesized from its three constituent amino acids by consecutive action of two cytosolic ATP-dependent enzymes: GCL and glutathione synthetase (17). GCL catalyzes the conjugation of the ␥-carboxyl group of L-glutamate to the amino group of L-cysteine (17). The proposed catalytic mechanism proceeds via phosphorylation of the ␥-carboxylate of L-glutamate by ATP (18 -20). The ␣-amino group of L-cysteine acts as a nucleophile, attacking the ␥-glutamyl phosphate intermediate to produce ␥-glutamylcysteine. This dipeptide is then coupled in an analogous fashion to glycine by glutathione synthetase to generate glutathione. As the committed step of glutathione biosynthesis, GCL activity is regulated by L-cysteine availability (21), feedback inhibition by glutathione (22), and transcriptional and post-translational regulation (23).Based on sequence analysis, three distinct groups of GCL have been identified. Groups 1 and 3 are comprised of bacterial and plant orthologues (24). Recently, x-ray crystal structures of Escherichia coli (Group 1) (25) and Brassica juncea (Group 3) (26) GCL were described, providing the first structural insights into ␥-glutamylc...

Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans

“…Yeasts share the same lineage as their protozoan and mammalian counterparts, but even so, significant differences exist between the human and the yeast enzymes. The mammalian enzyme is heterodimeric, with catalytic and regulatory subunits, while C. glabrata and C. albicans, like S. cerevisiae, seem to lack the regulatory subunit (Biterova & Barycki, 2009). C. albicans Gcs1p has a length of 772 amino acids and appears to contain a 90 amino acid insertion when compared with the sequences of C. glabrata and S. cerevisiae.…”

Structural Basis for Feedback and Pharmacological Inhibition of Saccharomyces cerevisiae Glutamate Cysteine Ligase

“…Crystals were grown at 18°C out of a solution of 12% (w/v) polyethylene glycol 400, 100 mM MES, pH 6.8, with the dimensions 0.15 ϫ 0.15 ϫ 0.15 mm, as described previously (24). Prior to data collection, the crystals were soaked in a stabilizing solution containing 30% polyethylene glycol 400 and the appropriate ligands and then vitrified in liquid nitrogen (26).…”

“…Protein Expression and Purification-ScGCL was expressed in Escherichia coli Rosetta TM 2(DE3) pLysS cells (Novagen) and purified to homogeneity as described previously (24). Briefly, soluble cell lysates were cleared of debris by centrifugation and ScGCL isolated by affinity chromatography using a HisTrap Chelating HP Column (GE Healthcare).…”

“…Recently, we reported the crystal structure of Saccharomyces cerevisiae GCL (ScGCL) in complex with L-glutamate, Mg 2ϩ , and ADP (24). As the first structure of a Group 2 glutamate cysteine ligase, examination of the model provided important molecular details of substrate recognition and led to the identification of key catalytic residues.…”

Expression of Bacterial GshF in Pichia pastoris for Glutathione Production

“…GshF also has other potential advantages over the conventional ␥-GCS/GS systems when used for GSH production. First, the feedback inhibition of ␥-GCS activity by GSH is considered a physiological mechanism to prevent the overaccumulation of GSH (1,2). GshF from S. agalactiae and Streptococcus thermophilus was shown to be insensitive to an increased concentration of GSH (9,11).…”