Trypanosoma brucei γ-Glutamylcysteine Synthetase

Glutamate-cysteine ligase (GCL) has a key influence on glutathione homeostasis. It has been proposed that mammalian GCL is regulated by the redox environment, and we show here that cysteine residues in the Drosophila melanogaster GCL modifier subunit (DmGCLM) can form covalent interactions with the catalytic subunit (DmGCLC) and modify its activity. Candidate components of intersubunit disulfides (Cys 213 , Cys 214 , and Cys 267 ) were identified using matrix-assisted laser desorption ionization time-of-flight spectroscopy of iodoacetamide-modified DmGCLM as well as examination of the evolutionary conservation of cysteines. Mutation of the 3 cysteine residues allowed DmGCLM to associate with DmGCLC, but inhibited the formation of intersubunit disulfides. This caused a 2-fold reduction in the catalytic efficiency of Drosophila GCL, although activity remained significantly higher than the catalytic subunit alone. The cysteine mutant was also more sensitive to inhibition by glutathione than the unmodified holoenzyme. Notably, human GCLM could substitute for DmGCLM in modification of DmGCLC activity. The role of DmGCLM in vivo was examined by analysis of a Drosophila mutant (l(3)L0580) containing a P-element insertion in Gclm. We found that the P-element is not responsible for the lethal phenotype and separated the recessive lethal mutation from the P-element by recombination. This yielded two fully viable and fertile recombinants bearing the P-element insertion, which Western and Northern blotting indicated is a severely hypomorphic allele of Gclm. Glutathione levels were ϳ2-fold lower in the Gclm L0580 mutants than in control strains, demonstrating the importance of DmGCLM in the regulation of glutathione homeostasis in vivo.The intracellular redox environment is of critical importance in cell physiology. It has a major influence on signaling pathways and cell fate in response to stress (1). Glutathione is one of the key influences on the redox state of the cell; and accordingly, intracellular glutathione levels are subject to multilateral regulatory mechanisms (2, 3).A major player in the regulation of glutathione homeostasis is glutamate-cysteine ligase (GCL), 1 which catalyzes the first and rate-limiting step in de novo synthesis of glutathione from precursor amino acids (4). GCL is located in the cytoplasm, and its activity appears to be regulated at several levels by the antioxidant status of the cell. In addition to being subject to transcriptional activation by pro-oxidants (5-7), evidence suggests that the catalytic activity is subject to redox regulation by the reversible formation of disulfide bridges between its two subunits (8, 9). Furthermore, GCL is subject to feedback inhibition by glutathione at concentrations that are physiologically relevant (10). The complex regulation of GCL activity highlights its pivotal role in controlling cellular glutathione synthesis.GCL is a heterodimer composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). The presence of GCLM modulates the catalytic prop...

Section: Discussionmentioning

confidence: 99%

The Modifier Subunit of Drosophila Glutamate-Cysteine Ligase Regulates Catalytic Activity by Covalent and Noncovalent Interactions and Influences Glutathione Homeostasis in Vivo

Fraser

Kansagra

Kotecki

et al. 2003

“…Protein was purified over a Ni 2ϩ -agarose column followed by gel filtration (22). Protein solutions were stored in 50 mM Tris, pH 8, 100 mM NaCl, 5 mM MgCl 2 at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Protein solutions were stored in 50 mM Tris, pH 8, 100 mM NaCl, 5 mM MgCl 2 at 4°C. Protein concentration was determined by absorbance at 280 nm with previously determined extinction coefficient (22).…”

Section: Methodsmentioning

confidence: 99%

“…A partial model of the ␥-GCS active site was constructed based on the GS structure allowing the Mg 2ϩ , MgATP, and L-Glu-binding sites to be modeled. This model of the ␥-GCS active site residues was used as a guide for experimental studies of the T. brucei ␥-GCS, which has previously been kinetically characterized (21,22). Based on the homology to GS, six conserved residues in ␥-GCS are predicted to form two metal-binding sites (n1: Glu-55, Glu-93, Glu-100; and n2: Glu-53, Glu-489, Gln-321).…”

Section: From the Departments Of ‡Pharmacology And ʈBiochemistry And mentioning

confidence: 99%

See 1 more Smart Citation

Structure Prediction and Active Site Analysis of the Metal Binding Determinants in γ-Glutamylcysteine Synthetase

Abbott¹,

Pei²,

Ford³

et al. 2001

Self Cite

␥-Glultamylcysteine synthetase (␥-GCS) catalyzes the first step in the de novo biosynthesis of glutathione. In trypanosomes, glutathione is conjugated to spermidine to form a unique cofactor termed trypanothione, an essential cofactor for the maintenance of redox balance in the cell. Using extensive similarity searches and sequence motif analysis we detected homology between ␥-GCS and glutamine synthetase (GS), allowing these proteins to be unified into a superfamily of carboxylateamine/ammonia ligases. The structure of ␥-GCS, which was previously poorly understood, was modeled using the known structure of GS. Two metal-binding sites, each ligated by three conserved active site residues (n1: Glu-55, Glu-93, Glu-100; and n2: Glu-53, Gln-321, and Glu-489), are predicted to form the catalytic center of the active site, where the n1 site is expected to bind free metal and the n2 site to interact with MgATP. To elucidate the roles of the metals and their ligands in catalysis, these six residues were mutated to alanine in the Trypanosoma brucei enzyme. All mutations caused a substantial loss of activity. Most notably, E93A was able to catalyze the L-Glu-dependent ATP hydrolysis but not the peptide bond ligation, suggesting that the n1 metal plays an important role in positioning L-Glu for the reaction chemistry. The apparent K m values for ATP were increased for both the E489A and Q321A mutant enzymes, consistent with a role for the n2 metal in ATP binding and phosphoryl transfer. Furthermore, the apparent K d values for activation of E489A and Q321A by free Mg 2؉ increased. Finally, substitution of Mn 2؉ for Mg 2؉ in the reaction rescued the catalytic deficits caused by both mutations, demonstrating that the nature of the metal ligands plays an important role in metal specificity.Glutathione (␥-glutamyl-cysteinyl-glycine) is a tripeptide thiol that acts as a sulfhydryl buffer. It is present in mammalian cells at high concentrations and plays an important role in many cellular processes, such as detoxification of oxidative species, protein and DNA synthesis, and cellular import of amino acids (1, 2). The parasitic protozoa Trypanosoma brucei causes African sleeping sickness, a disease responsible for significant morbidity and mortality in Africa. The identification of metabolic differences between parasite and host is an important step in the development of new anti-trypanosomal agents (3). Mammals and trypanosomes differ in their metabolism of glutathione. Trypanosomes utilize a unique molecule that is a conjugate of glutathione and spermidine, called trypanothione, to maintain reduced thiol pools in the cell (4). The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is the formation of ␥-glutamylcysteine, catalyzed by ␥-glutamylcysteine synthetase (␥-GCS) 1 (5). ␥-GCS is the rate-limiting enzyme in the synthesis of glutathione in mammalian cells (6) and of trypanothione in the trypanosomatid, Leishmania tarentolae (7).␥-GCS catalyzes the formation of a peptide bond between the ␥-car...

“…␥-GCS has been demonstrated to be the ratelimiting enzyme in the biosynthesis of GSH in mammalian cells (15) and of T(SH) 2 in Leishmania tarentolae (16). The enzyme from T. brucei has been biochemically characterized by study of the recombinant enzyme (17)(18)(19)(20).…”

mentioning

confidence: 99%

Gene Knockdown of γ-Glutamylcysteine Synthetase by RNAi in the Parasitic Protozoa Trypanosoma brucei Demonstrates That It Is an Essential Enzyme

Huynh

Harmon

et al. 2003

Self Cite

The parasitic protozoa Trypanosoma brucei utilizes a novel cofactor (trypanothione, T(SH) 2 ), which is a conjugate of GSH and spermidine, to maintain cellular redox balance. ␥-Glutamylcysteine synthetase (␥-GCS) catalyzes the first step in the biosynthesis of GSH. To evaluate the importance of thiol metabolism to the parasite, RNA i methods were used to knock down gene expression of ␥-GCS in procyclic T. brucei cells. Induction of ␥-GCS RNA i with tetracycline led to cell death within 4 -6 days post-induction. Cell death was preceded by the depletion of the ␥-GCS protein and RNA and by the loss of the cellular pools of GSH and T(SH) 2 . The addition of GSH (80 M) to cell cultures rescued the RNA i cell death phenotype and restored the intracellular thiol pools to wild-type levels. Treatment of cells with buthionine sulfoximine (BSO), an enzyme-activated inhibitor of ␥-GCS, also resulted in cell death. However, the toxicity of the inhibitor was not reversed by GSH, suggesting that BSO has more than one cellular target. BSO depletes intracellular thiols to a similar extent as ␥-GCS RNA i ; however, addition of GSH did not restore the pools of GSH and T(SH) 2 . These data suggest that BSO also acts to inhibit the transport of GSH or its peptide metabolites into the cell. The ability of BSO to inhibit both synthesis and transport of GSH likely makes it a more effective cytotoxic agent than an inhibitor with a single mode of action. Finally the potential for the T(SH) 2 biosynthetic enzymes to be regulated in response to reduced thiol levels was studied. The expression levels of ornithine decarboxylase and of S-adenosylmethionine decarboxylase, two essential enzymes in spermidine biosynthesis, remained constant in induced ␥-GCS RNA i cell lines.