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Structural studies of redox homeostasis enzymes

Ekaterina I Biterova, University of Nebraska - Lincoln


In mammalian organisms the intracellular redox status is maintained by two major thiol buffers: glutathione and thioredoxin. Glutathione is a critically important low molecular weight thiol responsible for the maintenance of intracellular redox homeostasis. Glutathione provides defense against oxidative or chemical stress and is an important cofactor in several biosynthetic and detoxification pathways. Thioredoxins are the major cellular protein disulfide reductases and are responsible for the regulation of numerous biochemical processes within the cell. ^ Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) catalyzes the ATP-dependent peptide bond formation between the γ-carboxylate of glutamate and the α-amino group of cysteine. The production of γ-glutamylcysteine is the first and rate-limiting step in the de novo biosynthesis of glutathione. We determined the structures of ScGCL in the presence of glutamate and MgCl2 (2.1 Å; R=18.2%, R free=21.9%), and in complex with ADP, glutamate, and MgCl2 (2.7 Å; R=18.0%, Rfree=24.4%). In addition, the structures of ScGCL in the presence of the feedback inhibitor, glutathione (2.5 Å; R = 19.6%; Rfree = 25.1%), and a pharmacological inhibitor, buthionine sulfoximine (BSO), ATP and MgCl2 (2.2 Å; R = 18.1%; Rfree = 23.9%) have been determined. Examination of the resulting structures provides unprecedented insight into the mechanistic details of group 2 glutamate cysteine ligases, and clarifies the Mg 2+ 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 the catalytic mechanism employed by ScGCL is conserved across the group 2 subfamily of γ-glutamate cysteine ligases. ^ Thioredoxin is maintained in a reduced state by thioredoxin reductase. Thioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. We determined the structure of mitochondrial TrxR, TrxR2. Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 Å; R = 22.2%; Rfree = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 Å; R = 24.1%; Rfree = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases.^

Subject Area

Chemistry, Biochemistry

Recommended Citation

Biterova, Ekaterina I, "Structural studies of redox homeostasis enzymes" (2009). ETD collection for University of Nebraska - Lincoln. AAI3350441.