Glutathione catalysis and the reaction mechanisms of glutathione-dependent enzymes

Background

Glutathione-dependent catalysis is a metabolic adaptation to chemical challenges encountered by all life forms. In the course of evolution, nature optimized numerous mechanisms to use glutathione as the most versatile nucleophile for the conversion of a plethora of sulfur-, oxygen- or carbon-containing electrophilic substances.

Scope of review

This comprehensive review summarizes fundamental principles of glutathione catalysis and compares the structures and mechanisms of glutathione-dependent enzymes, including glutathione reductase, glutaredoxins, glutathione peroxidases, peroxiredoxins, glyoxalases 1 and 2, glutathione transferases and MAPEG. Moreover, open mechanistic questions, evolutionary aspects and the physiological relevance of glutathione catalysis are discussed for each enzyme family.

Major conclusions

It is surprising how little is known about many glutathione-dependent enzymes, how often reaction geometries and acid–base catalysts are neglected, and how many mechanistic puzzles remain unsolved despite almost a century of research. On the one hand, several enzyme families with non-related protein folds recognize the glutathione moiety of their substrates. On the other hand, the thioredoxin fold is often used for glutathione catalysis. Ancient as well as recent structural changes of this fold did not only significantly alter the reaction mechanism, but also resulted in completely different protein functions.

General significance

Glutathione-dependent enzymes are excellent study objects for structure–function relationships and molecular evolution. Notably, in times of systems biology, the outcome of models on glutathione metabolism and redox regulation is more than questionable as long as fundamental enzyme properties are neither studied nor understood. Furthermore, several of the presented mechanisms could have implications for drug development. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Glutathione protects Lactococcus lactis against oxidative stress

Glutathione was found in several dairy Lactococcus lactis strains grown in M17 medium. None of these strains was able to synthesize glutathione. In chemically defined medium, L. lactis subsp. cremoris strain SK11 was able to accumulate up to approximately 60 mM glutathione when this compound was added to the medium. Stationary-phase cells of strain SK11 grown in chemically defined medium supplemented with glutathione showed significantly increased resistance (up to fivefold increased resistance) to treatment with H2O2 compared to the resistance of cells without intracellular glutathione. The resistance to H2O2 treatment was found to be dependent on the accumulation of glutathione in 16 strains of L. lactis tested. We propose that by taking up glutathione, L. lactis might activate a glutathione-glutathione peroxidase-glutathione reductase system in stationary-phase cells, which catalyzes the reduction of H2O2. Glutathione reductase, which reduces oxidized glutathione, was detectable in most strains of L. lactis, but the activities of different strains were very variable. In general, the glutathione reductase activities of L. lactis subsp. lactis are higher than those of L. lactis subsp. cremoris, and the activities were much higher when strains were grown aerobically. In addition, glutathione peroxidase is detectable in strain SK11, and the level was fivefold greater when the organism was grown aerobically than when the organism was grown anaerobically. Therefore, the presence of glutathione in L. lactis could result in greater stability under storage conditions and quicker growth upon inoculation, two important attributes of successful starter cultures.     

星形胶质细胞通过谷胱甘肽保护MN9D细胞抵抗鱼藤酮所致氧化应激

星形胶质细胞维持神经元微环境,给予营养和代谢支持,并调节其对损伤的反应。鱼藤酮特异阻断线粒体复合物Ⅰ,长期暴露于鱼藤酮可能增加患帕金森病的几率,并引起帕金森综合征。然而,星形胶质细胞在鱼藤酮所致多巴胺能神经元损伤过程中的作用尚无报道。本研究采用多巴胺能神经元细胞系MN9D细胞模型,将经过或未经过星形胶质细胞条件培养基处理的MN9D细胞暴露于不同浓度的鱼藤酮中,用计数法测生长曲线,MTT法测细胞活性,DCFH染色流式细胞仪测氧化应激水平,比色法测还原型谷胱甘肽含量。结果显示,MN9D细胞在条件和普通培养基培养条件下生长曲线无明显差别;鱼藤酮浓度依赖性地降低细胞活性;不同浓度鱼藤酮作用24、48h后,经条件培养基处理的细胞其活性显著高于普通培养基培养的细胞：不同浓度的条件培养基都有保护作用,纯的条件培养基保护作用稍弱：预先24h条件培养基处理或同时给予鱼藤酮和条件培养基处理都有保护作用,鱼藤酮作用12h后再给予条件培养基则无保护作用;经条件培养基处理的细胞氧化应激水平降低：另外,条件培养基提高了细胞内还原型谷胱甘肽含量,缓解了鱼藤酮所致的谷胱甘肽耗竭。结果提示,星形胶质细胞可保护MN9D细胞抵抗鱼藤酮所致的氧化应激,还原型谷胱甘肽可能参与了该保护过程。     

Glutathione metabolizing enzymes and oxidative stress in ferric nitrilotriacetate mediated hepatic injury

Summary

Glutathione (GSH) plays several important roles in the protection of cells against oxidative damage, particularly following exposure to xenobiotics. Ferric nitrilotriacetate (Fe-NTA) is a potent depletor of GSH and also enhances tissue lipid peroxidation. In this study, we show the effect of Fe-NTA treatment on hepatic GSH and some of the glutathione metabolizing enzymes, oxidant generation and liver damage. The level of hepatic GSH and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, and glucose 6-phosphate dehydrogenase all decrease following Fe-NTA administration. In these parameters the maximum decrease occurred at 12 h following Fe-NTA treatment. In contrast, γ-glutamyl transpeptidase was increased at this time. Not surprisingly, the increase in the activity of γ-glutamyl transpeptidase and decreases in GSH, glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase and glutathione S-transferase were found to be dependent on the dose of Fe-NTA administered. Fe-NTA administration also enhances the production of H₂O₂ and increases hepatic lipid peroxidation. Parallel to these changes, Fe-NTA enhances liver damage as evidenced by increases in serum transaminases. Once again, the liver damage is dependent on the dose of Fe-NTA and is maximal at 12 h. Pretreatment of animals with antioxidant, butylated hydroxy anisole (BHA), protects against Fe-NTA-mediated hepatotoxicity further supporting the involvement of oxidative stress in Fe-NTA-mediated hepatic damage. In aggregate, our results indicate that Fe-NTA administration eventuates in decreased hepatic GSH, a fall in the activities of glutathione metabolizing enzymes and excessive production of oxidants, all of which are involved in the cascade of events leading to iron-mediated hepatic injury.     

Glutathione S-transferase π localizes in mitochondria and protects against oxidative stress

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the protection of cellular components against anti-cancer drugs or peroxidative stress. Previously we found that GST π, an isoform of the GSTs, is transported into the nucleus. In the present study, we found that GST π is present in mitochondria as well as in the cytosol and nucleus in mammalian cell lines. A construct comprising the 84 amino acid residues in the amino-terminal region of GST π and green fluorescent protein was detected in the mitochondria. The mutation of arginine to alanine at positions 12, 14, 19, 71, and 75 in full-length GST π completely abrogated the ability to distribute in the mitochondria, suggesting that arginine, a positively charged residue, is required for the mitochondrial transport of GST π. Chemicals generating reactive oxygen species, such as rotenone and antimycin A, decreased cell viability and reduced mitochondrial membrane potential. The overexpression of GST π diminished these changes. GST π-targeting siRNA abolished the protective effect of GST π on the mitochondria under oxidative stress. The findings indicate that the peptide signal is conducive to the mitochondrial localization of GST π under steady-state conditions without alternative splicing or posttranslational modifications such as proteolysis, suggesting that GST π protects mitochondria against oxidative stress.     

A Sco homologue plays a role in defence against oxidative stress in pathogenic Neisseria

Sco proteins are found in mitochondria and in a variety of oxidase positive bacteria. Although Sco is required for the formation of the Cu(A) centre in a cytochrome oxidase of the aa(3) type, it was observed that oxidases with a Cu(A) centre are not present in many bacteria that contain a Sco homologue. Two bacteria of this type are the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. The sco genes of N. gonorrhoeae strain 1291 and N. meningitidis strain MC58 were cloned, inactivated by inserting a kanamycin resistance cassette and used to make knockout mutants by allelic exchange. Both N. gonorrhoeae and N. meningitidis sco mutants were highly sensitive to oxidative killing by paraquat, indicating that Sco is involved in protection against oxidative stress in these bacteria.     

Molecular basis of defence against oxidative stress in Entamoeba histolytica and Giardia lamblia

Gastrointestinal protozoa, viz. Entamoeba histolytica and Giardia, are able to survive in a microaerobic environment. The role of parasitic factors, particularly cysteine, cysteine-rich proteins, superoxide dismutase, and certain alternative mechanisms, has been described in the defence against oxidative stress. The role of the host-derived factors, particularly the phagocytosis of bacteria or host erythrocytes (intact or their enzymatic/non enzymatic components), in the detoxification of reactive oxygen metabolites in E. histolytica may provide novel approaches for the chemotherapy of invasive amoebiasis.     

Metabolic defence against oxidative stress: the road less travelled so far

Bacteria have survived, and many have thrived, since antiquity in the presence of the highly‐reactive chalcogen—oxygen (O₂). They are known to evoke intricate strategies to defend themselves from the reactive by‐products of oxygen—reactive oxygen species (ROS). Many of these detoxifying mechanisms have been extensively characterized; superoxide dismutase, catalases, alkyl hydroperoxide reductase and the glutathione (GSH)‐cycling system are responsible for neutralizing specific ROS. Meanwhile, a pool of NADPH—the reductive engine of many ROS‐combating enzymes—is maintained by metabolic enzymes including, but not exclusively, glucose‐6 phosphate dehydrogenase (G6PDH) and NADP‐dependent isocitrate dehydrogenase (ICDH‐NADP). So, it is not surprising that evidence continues to emerge demonstrating the pivotal role metabolism plays in mitigating ROS toxicity. Stemming from its ability to concurrently decrease the production of the pro‐oxidative metabolite, NADH, while augmenting the antioxidative metabolite, NADPH, metabolism is the fulcrum of cellular redox potential. In this review, we will discuss the mounting evidence positioning metabolism and metabolic shifts observed during oxidative stress, as critical strategies microbes utilize to thrive in environments that are rife with ROS. The contribution of ketoacids—moieties capable of non‐enzymatic decarboxylation in the presence of oxidants—as ROS scavengers will be elaborated alongside the metabolic pathways responsible for their homeostases. Further, the signalling role of the carboxylic acids generated following the ketoacid‐mediated detoxification of the ROS will be commented on within the context of oxidative stress.     

Azaphilones biosynthesis complements the defence mechanism of Trichoderma guizhouense against oxidative stress

Filamentous fungi are known as producers of a large array of diverse secondary metabolites (SMs) that aid in securing their environmental niche. Here, we demonstrated that the SMs have an additional role in fungal defence against other fungi: Trichoderma guizhouense, a mycoparasite, is able to antagonize Fusarium oxysporum f. sp. cubense race 4 (Foc4) by forming aerial hyphae that kill the host with hydrogen peroxide. At the same time, a gene cluster comprising two polyketide synthases is strongly expressed. Using functional genetics, we characterized this cluster and identified its products as azaphilones (termed as trigazaphilones). The trigazaphilones were found lacking of antifungal toxicity but exhibited high radical scavenging activities. The antioxidant property of trigazaphilones was in vivo functional under various tested conditions of oxidative stress. Thus, we conclude that the biosynthesis of trigazaphilones serves as a complementary antioxidant mechanism and defends T. guizhouense against the hydrogen peroxide that it produces to combat other fungi like Foc4.     

Glutathione, oxidative stress and neurodegeneration.

There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Friedreich's ataxia and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species and mitochondrial dysfunction. Here, we review the evidence for a disturbance of glutathione homeostasis that may either lead to or result from oxidative stress in neurodegenerative disorders. Glutathione is an important intracellular antioxidant that protects against a variety of different antioxidant species. An important role for glutathione was proposed for the pathogenesis of Parkinson's disease, because a decrease in total glutathione concentrations in the substantia nigra has been observed in preclinical stages, at a time at which other biochemical changes are not yet detectable. Because glutathione does not cross the blood-brain barrier other treatment options to increase brain concentrations of glutathione including glutathione analogs, mimetics or precursors are discussed.     

Glutathione S-transferase in the defence against pyrethroids in insects

The correlation between the natural levels of GST and the tolerance to the insecticide decamethrin (dMT), as well as the interaction between the molecules of affinity purified enzyme and the insecticide were investigated in order to collect further information on the obscure role of the Glutathione S-transferase system (GST) as a mechanism of defence against pyrethroids. The studies were carried out, comparatively, on the larvae and pupae developmental stages of the coleopteran Tenebrio molitor, which exhibit varying natural levels of GST activity. No stage dependent susceptibility of the insect against pyrethroid insecticides was found during the first 24 h, however 48 h after treatment, the KD50 dose increased significantly due to the recovery of some individuals from the larvae stage. Simultaneous injection of decamethrin with compounds which inhibit GST activity in vitro, resulted in an increased tolerance, which was more pronounced in the pupae stage. Inhibition studies combined with competitive fluorescence spectroscopy and high pressure liquid chromatography (HPLC) showed that the insecticide binds probably to the active site of the enzyme inhibiting its activity towards CDNB in a competitive manner, but is not conjugated with GSH. According to this, GST offers a passive protection towards pyrethroid insecticides by binding to their molecule in a sequestering mechanism.     

Glutathione contributes to plant defence against parasitic cyst nematodes

Cyst nematodes (CNs) are an important group of root-infecting sedentary endoparasites that severely damage many crop plants worldwide. An infective CN juvenile enters the host's roots and migrates towards the vascular cylinder, where it induces the formation of syncytial feeding cells, which nourish the CN throughout its parasitic stages. Here, we examined the role of glutathione (l -γ-glutamyl-l -cysteinyl-glycine) in Arabidopsis thaliana on infection with the CN Heterodera schachtii. Arabidopsis lines with mutations pad2, cad2, or zir1 in the glutamate–cysteine ligase (GSH1) gene, which encodes the first enzyme in the glutathione biosynthetic pathway, displayed enhanced CN susceptibility, but susceptibility was reduced for rax1, another GSH1 allele. Biochemical analysis revealed differentially altered thiol levels in these mutants that was independent of nematode infection. All glutathione-deficient mutants exhibited impaired activation of defence marker genes as well as genes for biosynthesis of the antimicrobial compound camalexin early in infection. Further analysis revealed a link between glutathione-mediated plant resistance to CN infection and the production of camalexin on nematode infection. These results suggest that glutathione levels affect plant resistance to CN by fine-tuning the balance between the cellular redox environment and the production of compounds related to defence against infection.     

Glutathione and p53 independently mediate responses against oxidative stress in ES cells.

We have investigated the roles of the antioxidant glutathione and p53 in the response of embryonic stem (ES) cells to oxidative stress. ES cells express gammaGCS, a critical enzyme in glutathione (GSH) biosynthesis. Treatment with the pro-oxidant menadione led to elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO, a specific gammaGCS inhibitor depleted GSH pools and prevented the menadione-induced increase in GSH, sensitizing cells to oxidative insult. Although p53 status had no bearing on either the basal levels of GSH or the menadione-induced GSH response, the levels of menadione-induced apoptosis were reduced in the absence of p53. We conclude that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage. Furthermore, the presence of an intact p53 pathway confers a long-term growth advantage post oxidative stress. Thus, in the absence of p53 ES cells bearing genotoxic damage are less likely to be propagated, suggesting that p53-dependent apoptosis acts to limit the deleterious effects of oxidative stress during early development.     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Role of Acinetobacter calcoaceticus 3,4-dihydrocoumarin hydrolase in oxidative stress defence against peroxoacids.

The physiological role of a bifunctional enzyme, 3,4-dihydrocoumarin hydrolase (DCH), which is capable of both hydrolysis of ester bonds and organic acid-assisted bromination of organic compounds, was investigated. Purified DCH from Acinetobacter calcoaceticus F46 catalysed dose- and time-dependent degradation of peracetic acid. The gene (dch) was cloned from the chromosomal DNA of the bacterium. The dch ORF was 831 bp long, corresponding to a protein of 272 amino acid residues, and the deduced amino acid sequence showed high similarity to those of bacterial serine esterases and perhydrolases. The dch gene was disrupted by homologous recombination on the A. calcoaceticus genome. The dch disruptant strain was more sensitive to growth inhibition by peracetic acid than the parent strain. On the other hand, the recombinant Escherichia coli cells expressing dch were more resistant to peracetic acid. A putative catalase gene was found immediately downstream of dch, and Northern blot hybridization analysis revealed that they are transcribed as part of a polycistronic mRNA. These results suggested that in vivo DCH detoxifies peroxoacids in conjunction with the catalase, i.e. peroxoacids are first hydrolysed to the corresponding acids and hydrogen peroxide by DCH, and then the resulting hydrogen peroxide is degraded by the catalase.     

Copper-induced oxidative stress and antioxidant defence in Arabidopsis thaliana

Content of reactive oxygen species (ROS): O2*-, H2O2 and OH* as well as activities of antioxidant enzymes: superoxide dismutase (SOD), guaiacol peroxidase (POX) and catalase (CAT) were studied in leaves of Arabidopsis thaliana ecotype Columbia, treated with Cu excess (0, 5, 25, 30, 50, 75, 100, 150 and 300 microM). After 7 days of Cu action ROS content and the activity of SOD and POX increased, while CAT activity decreased in comparison with control. Activities of SOD, POX and CAT were correlated both with Cu concentration (0-75 microM) in the growth medium and with OH* content in leaves. Close correlation was also found between OH* content and Cu concentration. Oxidative stress in A. thaliana under Cu treatment expressed in elevated content of O2*-, H2O2 and OH* in leaves. To overcome it very active the dismutase- and peroxidase-related (and not catalase-related, as in other plants) ROS scavenging system operated in A. thaliana. Visual symptoms of phytotoxicity: chlorosis, necrosis and violet colouring of leaves as well as a reduction of shoot biomass occurred in plants.     

Thiol dependent oxidation of enzymes: The last chance against oxidative stress

• 1.1. A survey of known effects of oxidized thiols on enzyme activity reveals a potential concerted action on metabolic pathways determining an impairment of anabolic reduction processes and an activation of the oxidative arm of the hexose monophosphate shunt. Thus it appears that, following oxidative stress, the increase of disulphides may act in restoring a reduced state in the cell by specifically channelling the metabolic energy flux.

     

Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2-Cys peroxiredoxin

In general, oxidative stress, the consequence of an aerobic lifestyle, induces bacterial antioxidant defence enzymes. Here we report on a peroxiredoxin of Rhizobium etli, prxS, strongly expressed under microaerobic conditions and during the symbiotic interaction with Phaseolus vulgaris. The microaerobic induction of the prxS-rpoN2 operon is mediated by the alternative sigma factor RpoN and the enhancer-binding protein NifA. The RpoN-dependent promoter is also active under low-nitrogen conditions through the enhancer-binding protein NtrC. An additional symbiosis-specific weak promoter is located between prxS and rpoN2. Constitutive expression of prxS confers enhanced survival and growth to R. etli in the presence of H2O2. Single prxS mutants are not affected in their symbiotic abilities or defence response against oxidative stress under free-living conditions. In contrast, a prxS katG double mutant has a significantly reduced (>40%) nitrogen fixation capacity, suggesting a functional redundancy between PrxS and KatG, a bifunctional catalase-peroxidase. In vitro assays demonstrate the reduction of PrxS protein by DTT and thioredoxin. PrxS displays substrate specificity towards H2O2 (Km = 62 microM) over alkyl hydroperoxides (Km > 1 mM). Peroxidase activity is abolished in both the peroxidatic (C56) and resolving (C156) cysteine PrxS mutants, while the conserved C81 residue is required for proper folding of the protein. Resolving of the R. etli PrxS peroxidatic cysteine is probably an intramolecular process and intra- and intersubunit associations were observed. Taken together, our data support, for the first time, a role for an atypical 2-Cys peroxiredoxin against oxidative stress in R. etli bacteroids.     

Hexanoic acid protects tomato plants against Botrytis cinerea by priming defence responses and reducing oxidative stress

Treatment with the resistance priming inducer hexanoic acid (Hx) protects tomato plants from Botrytis cinerea by activating defence responses. To investigate the molecular mechanisms underlying hexanoic acid‐induced resistance (Hx‐IR), we compared the expression profiles of three different conditions: Botrytis‐infected plants (Inf), Hx‐treated plants (Hx) and Hx‐treated + infected plants (Hx+Inf). The microarray analysis at 24 h post‐inoculation showed that Hx and Hx+Inf plants exhibited the differential expression and priming of many Botrytis‐induced genes. Interestingly, we found that the activation by Hx of other genes was not altered by the fungus at this time point. These genes may be considered to be specific targets of the Hx priming effect and may help to elucidate its mechanisms of action. It is noteworthy that, in Hx and Hx+Inf plants, there was up‐regulation of proteinase inhibitor genes, DNA‐binding factors, enzymes involved in plant hormone signalling and synthesis, and, remarkably, the genes involved in oxidative stress. Given the relevance of the oxidative burst occurring in plant–pathogen interactions, the effect of Hx on this process was studied in depth. We showed by specific staining that reactive oxygen species (ROS) accumulation in Hx+Inf plants was reduced and more restricted around infection sites. In addition, these plants showed higher ratios of reduced to oxidized glutathione and ascorbate, and normal levels of antioxidant activities. The results obtained indicate that Hx protects tomato plants from B. cinerea by regulating and priming Botrytis‐specific and non‐specific genes, preventing the harmful effects of oxidative stress produced by infection.