ChaC1 upregulation reflects poor prognosis in a variety of cancers: analysis of the major missense SNPs of ChaC1 as an aid to refining prognosis.

The ChaC1 enzyme that catalyzes cytosolic glutathione degradation is highly upregulated in several cancers. In a systematic review of gene signature panels for cancer prognosis based on oxidative stress and ferroptosis genes, we observed that ChaC1 was found in panels in a wide variety of different cancers, with the upregulation correlating with poor prognosis. Since SNPs can have an impact on functionality and prognosis, ChaC1 SNPs from various databases were also investigated. Six frequently observed missense SNPs were chosen for reconstruction, and their functionality was evaluated. Three out of six SNPs resulted in either a partial or complete loss of ChaC1 function, and these SNPs had the changes R72Q, A156V, and G173S in their proteins. This study highlights the importance of ChaC1 in cancer prognosis across a wide variety of cancers. Additionally, the information on the SNPs of ChaC1 with altered enzymatic activities would improve the prognostic ability of these panels and facilitate treatment regimens.

Early discoveries on enzyme deficiencies in lysosomal storage diseases: The Indian contribution.

The discovery of enzyme deficiencies in lysosomal storage disorders began with two discoveries made in 1963. One of these was made by a Belgian scientist, Henri-Gery Hers, who discovered that in Pompe's disease there was a deficiency in α-glucosidase. The other was made by an international collaboration involving an American neurologist, James Austin, and an Indian biochemist, Bimal Bachhawat, where the enzyme arylsulfatase A was found deficient in metachromatic leukodystrophy. This article attempts to trace the events that led to this fruitful collaboration and how these two young investigators eventually discovered the defective enzyme in metachromatic leukodystrophy.

Cystinosis: Status of research and treatment in India and the world.

Cystinosis is an autosomally inherited rare genetic disorder in which cystine accumulates in the lysosome. The defect arises from a mutation in the lysosomal efflux pump, cystinosin (or CTNS). Despite the disease being known for more than a century, research, diagnosis, and treatment in India have been very minimal. In recent years, however, some research on cystinosis has been carried out on understanding the pathophysiology and in the development of a humanized yeast model for interrogating the CTNS protein. There has also been a greater awareness of the disease that has been facilitated by the formation of the Cystinosis Foundation of India just over a decade ago. Awareness among primary physicians is critical for early diagnosis, which in turn is critical for proper treatment. Eight different mutations have been observed in cystinosis patients in India, and the mutation spectrum seems different to what has been seen in the US and Europe. Despite these positive developments, there are immense hurdles still to be surmounted. This includes ensuring that the diagnosis is done sooner, making cysteamine more easily available, and, also for the future, to make accessible the promise of gene therapy to cystinosis patients.

The ChaC1 active site: Defining the residues and determining the role of ChaC1-exclusive residues in the structural and functional stability.

The glutathione degrading enzyme ChaC1 is highly upregulated in several cancers and viral infections making it a potential pharmacological target for cancer therapy. As an enzyme, however, ChaC1 has a relatively high Km (~2 mM) towards its natural substrate, and therefore finding its inhibitors becomes very difficult. Given this limitation, a careful mapping of the active site has become necessary. In the current study, the enzyme-substrate complex was generated by docking glutathione with the modeled hChaC1 structure. Using a combination of in silico and wet lab approaches, the active site residues forming direct interactions with the substrate glutathione were identified and validated. Furthermore, the role of residues exclusively conserved in the ChaC family and forming the surface of the active site were also explored for their putative role in active site stabilization. Mutants of these residues have been analysed for their structural stability and interaction with the substrate through MD simulations and MMGBSA binding energy calculations. These findings were experimentally validated by assessment of their function through in vivo assays in yeast. The experimental evidences along with the molecular modeling suggest that residues 38'YGSL'41, D68, R72, E115, and Y143 are responsible for high affinity binding of hChaC1 with the substrate/inhibitor, whereas the residues exclusive to the ChaC family are required for the structural stability of the enzyme and its active site. Such a characterization of essential active site and conserved residues is significant as a key step toward rational design of novel inhibitors of the ChaC1 enzyme.

Microbial electrosynthesis from carbon dioxide feedstock linked to yeast growth for the production of high-value isoprenoids.

The difficulty in producing multi-carbon and thus high-value chemicals from CO2 is one of the key challenges of microbial electrosynthesis (MES) and other CO2 utilization technologies. Here, we demonstrate a two-stage bioproduction approach to produce terpenoids (>C20) and yeast biomass from CO2 by linking MES and yeast cultivation approaches. In the first stage, CO2 (C1) is converted to acetate (C2) using Clostridium ljungdahlii via MES. The acetate is then directly used as the feedstock to produce sclareol (C20), ß-carotene (C40), and yeast biomass using Saccharomyces cerevisiae in the second stage. With the unpurified acetate-containing (1.5 g/L) spent medium from MES reactors, S. cerevisiae produced 0.32 ± 0.04 mg/L ß-carotene, 2.54 ± 0.91 mg/L sclareol, and 369.66 ± 41.67 mg/L biomass. The primary economic analysis suggests that sclareol and biomass production is feasible using recombinant S. cerevisiae and non-recombinant S. cerevisiae, respectively, directly from unpurified acetate-containing spent medium of MES.

A novel leishmanial copper P-type ATPase plays a vital role in parasite infection and intracellular survival.

Copper (Cu) is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of Cu are known to be utilized by host species against various pathogenic invasions. Leishmania, in both free-living and intracellular forms, exhibits appreciable tolerance toward Cu stress. While determining the mechanism of Cu-stress evasion employed by Leishmania, we identified and characterized a hitherto unknown Cu-ATPase in Leishmania major and established its role in parasite survival in host macrophages. This novel L. major Cu-ATPase, LmATP7, exhibits homology with its orthologs at multiple motifs. In promastigotes, LmATP7 primarily localized at the plasma membrane. We also show that LmATP7 exhibits Cu-dependent expression patterns and complements Cu transport in a Cu-ATPase-deficient yeast strain. Promastigotes overexpressing LmATP7 exhibited higher survival upon Cu stress, indicating efficacious Cu export compared with Wt and heterozygous LmATP7 knockout parasites. We further explored macrophage-Leishmania interactions with respect to Cu stress. We found that Leishmania infection triggers upregulation of major mammalian Cu exporter, ATP7A, in macrophages, and trafficking of ATP7A from the trans-Golgi network to endolysosomes in macrophages harboring amastigotes. Simultaneously, in Leishmania, we observed a multifold increase in LmATP7 transcripts as the promastigote becomes established in macrophages and morphs to the amastigote form. Finally, overexpressing LmATP7 in parasites increases amastigote survivability within macrophages, whereas knocking it down reduces survivability drastically. Mice injected in their footpads with an LmATP7-overexpressing strain showed significantly larger lesions and higher amastigote loads as compared with controls and knockouts. These data establish the role of LmATP7 in parasite infectivity and intramacrophagic survivability.

Heart failure and the glutathione cycle: an integrated view.

Heart failure results from the heart's inability to carryout ventricular contraction and relaxation, and has now become a worldwide problem. During the onset of heart failure, several signatures are observed in cardiomyocytes that includes fetal reprogramming of gene expression where adult genes are repressed and fetal genes turned on, endoplasmic reticulum stress and oxidative stress. In this short review and analysis, we examine these different phenomenon from the viewpoint of the glutathione cycle and the role of the recently discovered Chac1 enzyme. Chac1, which belongs to the family of γ-glutamylcyclotransferases, is a recently discovered member of the glutathione cycle, being involved in the cytosolic degradation of glutathione. This enzyme is induced during the Endoplasmic Stress response, but also in the developing heart. Owing to its exclusive action on reduced glutathione, its induction leads to an increase in the oxidative redox potential of the cell that also serves as signaling mechanism for calcium ions channel activation. The end product of Chac1 action is 5-oxoproline, and studies with 5-oxoprolinase (OPLAH), an enzyme of the glutathione cycle has revealed that down-regulation of OPLAH can lead to the accumulation of 5-oxproline which is an important factor in heart failure. With these recent findings, we have re-examined the roles and regulation of the enzymes in the glutathione cycle which are central to these responses. We present an integrated view of the glutathione cycle in the cellular response to heart failure.

An inquiry-based approach in large undergraduate labs: Learning, by doing it the "wrong" way.

Undergraduate laboratory courses, owing to their larger sizes and shorter time slots, are often conducted in highly structured modes. However, this approach is known to interfere with students' engagement in the experiments. To enhance students' engagement, we propose an alternative mode of running laboratory courses by creating some "disorder" in a previously adopted structure. After performing an experiment in the right way, the students were asked to repeat the experiment but with a variation at certain steps leading to the experiment being done the "wrong" way. Although this approach led to fewer experiments being conducted in a semester, it significantly enhanced the students' involvement. This was also reflected in the students' feedback. The majority of students preferred repeating an experiment with a variant protocol than performing a new experiment. Although we have tested this inquiry-based approach only for an undergraduate laboratory course in molecular biology, we believe such an approach could also be extended to undergraduate laboratory courses of other subjects.

A Genetic Screen for the Isolation of Mutants with Increased Flux in the Isoprenoid Pathway of Yeast.

The yeast Saccharomyces cerevisiae is one of the preferred hosts for the production of terpenoids through metabolic engineering. A genetic screen to identify novel mutants that can increase the flux in the isoprenoid pathway has been lacking. We present here the method that has led to the development of a carotenoid based visual screen by exploiting the carotenogenic genes from the red yeast Rhodosporidium toruloides, an organism known to have high levels of carotenoids. We also discuss the methods to use this screen for the identification of mutants that can lead to higher isoprenoid flux. The carotenoid based screen was developed in S. cerevisiae using phytoene synthase RtPSY1 and a hyperactive mutant of the enzyme phytoene dehydrogenase, RtCRTI(A393T) from Rhodosporidium toruloides. As validation of the genetic screen is critical at all stages, we describe the method to validate the screen using a known flux increasing gene, a truncated HMG1 (tHMG1). To demonstrate how this screen can be exploited to isolate mutants, we described how targeted mutagenesis of candidate gene, SPT15 a TATA binding protein involved in the global transcription machinery can be carried out to yield novel mutants with increased metabolic flux. Since it is also important to ensure that the isolated mutants are enhancing general isoprenoid flux, we describe how this can be established using an alternate isoprenoid, α-farnesene.

Role of phosphate limitation and pyruvate decarboxylase in rewiring of the metabolic network for increasing flux towards isoprenoid pathway in a TATA binding protein mutant of Saccharomyces cerevisiae.

BACKGROUND: Production of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several attempts have been made to reroute metabolic flux towards isoprenoid pathway in yeast. Most approaches have focused on the core isoprenoid pathway as well as on meeting the increased precursors and cofactor requirements. To identify unexplored genetic targets that positively influence the isoprenoid pathway activity, a carotenoid based genetic screen was previously developed and three novel mutants of a global TATA binding protein SPT15 was isolated for heightened isoprenoid flux in Saccharomyces cerevisiae. RESULTS: In this study, we investigated how one of the three spt15 mutants, spt15_Ala101Thr, was leading to enhanced isoprenoid pathway flux in S. cerevisiae. Metabolic flux analysis of the spt15_Ala101Thr mutant initially revealed a rerouting of the central carbon metabolism for the production of the precursor acetyl-CoA through activation of pyruvate-acetaldehyde-acetate cycle in the cytoplasm due to high flux in the reaction caused by pyruvate decarboxylase (PDC). This led to alternate routes of cytosolic NADPH generation, increased mitochondrial ATP production and phosphate demand in the mutant strain. Comparison of the transcriptomics of the spt15_Ala101Thr mutant cell with SPT15WT bearing cells shows upregulation of phosphate mobilization genes and pyruvate decarboxylase 6 (PDC6). Increasing the extracellular phosphate led to an increase in the growth rate and biomass but diverted flux away from the isoprenoid pathway. PDC6 is also shown to play a critical role in isoprenoid pathway flux under phosphate limitation conditions. CONCLUSION: The study not only proposes a probable mechanism as to how a spt15_Ala101Thr mutant (a global TATA binding protein mutant) could increase flux towards the isoprenoid pathway, but also PDC as a new route of metabolic manipulation for increasing the isoprenoid flux in yeast.

Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues.

Cch1p, the yeast homolog of the pore-forming subunit α1 of the mammalian voltage-gated Ca2+ channel (VGCC), is located on the plasma membrane and mediates the redox-dependent influx of Ca2+ Cch1p is known to undergo both rapid activation (after oxidative stress and or a change to high pH) and slow activation (after ER stress and mating pheromone activation), but the mechanism of activation is not known. We demonstrate here that both the fast activation (exposure to pH 8-8.5 or treatment with H2O2) and the slow activation (treatment with tunicamycin or α-factor) are mediated through a common redox-dependent mechanism. Furthermore, through mutational analysis of all 18 exposed cysteine residues in the Cch1p protein, we show that the four mutants C587A, C606A, C636A and C642A, which are clustered together in a common cytoplasmic loop region, were functionally defective for both fast and slow activations, and also showed reduced glutathionylation. These four cysteine residues are also conserved across phyla, suggesting a conserved mechanism of activation. Investigations into the enzymes involved in the activation reveal that the yeast glutathione S-transferase Gtt1p is involved in the glutathionylation of Cch1p, while the thioredoxin Trx2p plays a role in the Cch1p deglutathionylation.

Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines.

Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward-facing channels: the plasma membrane, Cch1p, and the vacuolar calcium channel, Yvc1p. Deletion of these channels partially rescues cells from glutathione depletion-induced cell death. Subsequent investigations on the Yvc1p channel, a homologue of the mammalian TRP channels, revealed that the channel is activated by glutathionylation. Yvc1p has nine cysteine residues, of which eight are located in the cytoplasmic regions and one on the transmembrane domain. We show that three of these cysteines, Cys-17, Cys-79, and Cys-191, are specifically glutathionylated. Mutation of these cysteines to alanine leads to a loss in glutathionylation and a concomitant loss in calcium channel activity. We further investigated the mechanism of glutathionylation and demonstrate a role for the yeast glutathione S-transferase Gtt1p in glutathionylation. Yvc1p is also deglutathionylated, and this was found to be mediated by the yeast thioredoxin, Trx2p. A model for redox activation and deactivation of the yeast Yvc1p channel is presented.

Charged/Polar-residue scanning of the hydrophobic face of transmembrane domain 9 of the yeast glutathione transporter, hgt1p, reveals a conformationally critical region for substrate transport.

Unraveling the mechanistic workings of membrane transporters has remained a challenging task. We describe a novel strategy that involves subjecting the residues of the hydrophobic face of a transmembrane helix to a charged/polar scanning mutagenesis. TMD9 of the yeast glutathione transporter, Hgt1p, has been identified as being important in substrate binding, and two residues, F523 and Q526, are expected to line the substrate translocation channel while the other face is hydrophobic. The hydrophobic face of TMD9 helix consists of residues A509, V513, L517, L520, I524, and I528, and these were mutated to lysine, glutamine, and glutamic acid. Among the 16 charged mutants created, six were nonfunctional, revealing a surprising tolerance of charged residues in the hydrophobic part of TM helices. Furthermore, the only position that did not tolerate any charged residue was I524, proximal to the substrate binding residues. However, P525, also proximal to the substrate binding residues, did tolerate charged/polar residues, suggesting that mere proximity to the substrate binding residues was not the only factor. The I524K/E/Q mutants expressed well and localized correctly despite lacking any glutathione uptake capability. Isolation of suppressors for all nonfunctional mutants yielded second-site suppressors only for I524K and I524Q, and suppressors for these mutations appeared at G202K/I and G202K/Q, respectively. G202 is in the hydrophilic loop between TMD3 and TMD4. The results suggest that I524 in the hydrophobic face interacts with this region and is also in a conformationally critical region for substrate translocation.

Defining the cytosolic pathway of glutathione degradation in Arabidopsis thaliana: role of the ChaC/GCG family of γ-glutamyl cyclotransferases as glutathione-degrading enzymes and AtLAP1 as the Cys-Gly peptidase.

Glutathione homoeostasis is critical to plant life and its adaptation to stress. The γ-glutamyl cycle of glutathione biosynthesis and degradation plays a pre-eminent role in glutathione homoeostasis. The genes encoding two enzymatic steps of glutathione degradation, the γ-glutamyl cyclotransferase (GGCT; acting on γ-glutamyl amino acids) and the Cys-Gly dipeptidase, have, however, lacked identification. We have investigated the family of GGCTs in Arabidopsis thaliana. We show through in vivo functional assays in yeast that all three members of the ChaC/GCG subfamily show significant activity towards glutathione but no detectable activity towards γ-glutamyl methionine. Biochemical characterization of the purified recombinant enzymes GGCT2;2 and GGCT2;3 further confirmed that they act specifically to degrade glutathione to yield 5-oxoproline and Cys-Gly peptide and show no significant activity towards γ-glutamyl cysteine. The Km for glutathione was 1.7 and 4.96 mM for GGCT2;2 and GGCT2;3 respectively and was physiologically relevant. Evaluation of representative members of other subfamilies indicates the absence of GGCTs from plants showing significant activity towards γ-glutamyl-amino acids as envisaged in the classical γ-glutamyl cycle. To identify the Cys-Gly peptidase, we evaluated leucine aminopeptidases (LAPs) as candidate enzymes. The cytosolic AtLAP1 (A. thaliana leucine aminopeptidase 1) and the putative chloroplastic AtLAP3 displayed activity towards Cys-Gly peptide through in vivo functional assays in yeast. Biochemical characterization of the in vitro purified hexameric AtLAP1 enzyme revealed a Km for Cys-Gly of 1.3 mM that was physiologically relevant and indicated that AtLAP1 represents a cytosolic Cys-Gly peptidase activity of A. thaliana. The studies provide new insights into the functioning of the γ-glutamyl cycle in plants.

New insights into the genetics of 5-oxoprolinase deficiency and further evidence that it is a benign biochemical condition.

UNLABELLED: Inherited 5-oxoprolinase (OPLAH) deficiency is a rare inborn condition characterised by 5-oxoprolinuria. To date, three OPLAH mutations have been described: p.H870Pfs in a homozygous state, which results in a truncated protein, was reported in two siblings, and two heterozygous missense changes, p.S323R and p.V1089I, were independently identified in two unrelated patients. We describe the clinical context of a young girl who manifested 5-oxoprolinuria together with dusky episodes and who is compound heterozygote for two novel OPLAH variations: p.G860R and p.D1241V. To gain insight into the aetiology of the 5-oxoprolinase deficiency, we investigated the pathogenicity of all the reported missense mutations in the OPLAH gene. A yeast in vivo growth assay revealed that only p.S323R, p.G860R and p.D1241V affected the activity of the enzyme. CONCLUSION: Taken together, this report further suggests that hereditary 5-oxoprolinase deficiency is a benign biochemical condition caused by mutations in the OPLAH gene, which are transmitted in an autosomal recessive manner, but 5-oxoprolinuria may be a chance association in other disorders.

Mutations in the N-terminal region of the Schizosaccharomyces pombe glutathione transporter pgt1⁺ allows functional expression in Saccharomyces cerevisiae.

Pgt1p encodes a glutathione transporter in Schizosaccharomyces pombe, orthologous to the Saccharomyces cerevisiae glutathione transporter, Hgt1p. Despite high similarity to Hgt1p, Pgt1p failed to display functionality during heterologous expression in S. cerevisiae. In the present study we employed a genetic strategy to investigate the reason behind the non-functionality of pgt1⁺ in S. cerevisiae. Functional mutants were isolated after in vitro mutagenesis. Several mutants were obtained and four mutants analysed. Among these, three yielded different point mutations in the N-terminal region (301-350 bp) of the transporter before the first transmembrane domain, while one mutant contained a deletion of 42 nucleotides within the same region. The mutant pgt1⁺ proteins not only expressed and localized correctly, but displayed high-affinity glutathione transport capabilities in S. cerevisae. Comparison of wild-type pgt1⁺ with the functional mutants revealed that a loss in protein expression was responsible for lack of functionality of wild-type pgt1⁺ in S. cerevisiae. The mRNA levels in wild-type and mutants were comparable, suggesting that the block was in translation. The formation of a strong stem-loop structure appeared to be responsible for inefficient translation in pgt1⁺ and disruption of these structures in the mutants was probably permitting translation. This was confirmed by making silent mutations in this region of wild-type pgt1⁺, which led to their functionality in S. cerevisiae. This genetic strategy to relieve functional blocks in expression should greatly facilitate the study of these and other transporters from more intractable genetic organisms in a heterologous expression system.

Glutathione transporters.

BACKGROUND: Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular milieu. GSH is also imported into the cytoplasm. The transports of glutathione across these different membranes in different systems have been biochemically demonstrated. However the molecular identity of the transporters has been established only in a few cases. SCOPE OF REVIEW: An attempt has been made to present the current state of knowledge of glutathione transporters from different organisms as well as different organelles. These include the most well characterized transporters, the yeast high-affinity, high-specificity glutathione transporters involved in import into the cytoplasm, and the mammalian MRP proteins involved in low affinity glutathione efflux from the cytoplasm. Other glutathione transporters that have been described either with direct or indirect evidences are also discussed. MAJOR CONCLUSIONS: The molecular identity of a few glutathione transporters has been unambiguously established but there is a need to identify the transporters of other systems and organelles. There is a lack of direct evidence establishing transport by suggested transporters in many cases. Studies with the high affinity transporters have led to important structure-function insights. GENERAL SIGNIFICANCE: An understanding of glutathione transporters is critical to our understanding of redox homeostasis in living cells. By presenting our current state of understanding and the gaps in our knowledge the review hopes to stimulate research in these fields. This article is part of a Special Issue entitled Cellular functions of glutathione.

Redox-dependent stability of the γ-glutamylcysteine synthetase enzyme of Escherichia coli: a novel means of redox regulation.

Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalysed by γ-GCS (γ-glutamylcysteine synthetase). The crystal structure of Escherichia coli γ-GCS has revealed the presence of a disulfide bond. As the disulfide-bonding cysteine residues Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and other cysteine residues. In a cysteine-free γ-GCS that was non-functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulfide bond, and one mutant that lacked any cysteine residues. Kinetic analysis of the wild-type and mutant enzymes revealed that the disulfide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulfide bond show a decreased half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.

Glutathione degradation by the alternative pathway (DUG pathway) in Saccharomyces cerevisiae is initiated by (Dug2p-Dug3p)2 complex, a novel glutamine amidotransferase (GATase) enzyme acting on glutathione.

The recently identified, fungi-specific alternative pathway of glutathione degradation requires the participation of three genes, DUG1, DUG2, and DUG3. Dug1p has earlier been shown to function as a Cys-Gly-specific dipeptidase. In the present study, we describe the characterization of Dug2p and Dug3p. Dug3p has a functional glutamine amidotransferase (GATase) II domain that is catalytically important for glutathione degradation as demonstrated through mutational analysis. Dug2p, which has an N-terminal WD40 and a C-terminal M20A peptidase domain, has no peptidase activity. The previously demonstrated Dug2p-Dug3p interaction was found to be mediated through the WD40 domain of Dug2p. Dug2p was also shown to be able to homodimerize, and this was mediated by its M20A peptidase domain. In vitro reconstitution assays revealed that Dug2p and Dug3p were required together for the cleavage of glutathione into glutamate and Cys-Gly. Purification through gel filtration chromatography confirmed the formation of a Dug2p-Dug3p complex. The functional complex had a molecular weight that corresponded to (Dug2p-Dug3p)(2) in addition to higher molecular weight oligomers and displayed Michaelis-Menten kinetics. (Dug2p-Dug3p)(2) had a K(m) for glutathione of 1.2 mm, suggesting a novel GATase enzyme that acted on glutathione. Dug1p activity in glutathione degradation was found to be restricted to its Cys-Gly peptidase activity, which functioned downstream of the (Dug2p-Dug3p)(2) GATase. The DUG2 and DUG3 genes, but not DUG1, were derepressed by sulfur limitation. Based on these studies and the functioning of GATases, a mechanism is proposed for the functioning of the Dug proteins in the degradation of glutathione.