Characterization of mitochondrial thioredoxin reductase from C. elegans

Thioredoxin reductase catalyzes the NADPH-dependent reduction of the catalytic disulfide bond of thioredoxin. In mammals and other higher eukaryotes, thioredoxin reductases contain the rare amino acid selenocysteine at the active site. The mitochondrial enzyme from Caenorhabditis elegans, however, contains a cysteine residue in place of selenocysteine. The mitochondrial C. elegans thioredoxin reductase was cloned from an expressed sequence tag and then produced in Escherichia coli as an intein-fusion protein. The purified recombinant enzyme has a kcat of 610 min(-1) and a Km of 610 microM using E. coli thioredoxin as substrate. The reported kcat is 25% of the kcat of the mammalian enzyme and is 43-fold higher than a cysteine mutant of mammalian thioredoxin reductase. The enzyme would reduce selenocysteine, but not hydrogen peroxide or insulin. The flanking glycine residues of the GCCG motif were mutated to serine. The mutants improved substrate binding, but decreased the catalytic rate.     

Mitochondrial thioredoxin reductase in bovine adrenal cortex its purification, properties, nucleotide/amino acid sequences, and identification of selenocysteine.

Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.     

Molecular Genetics Evidence for the in Vivo Roles of the Two Major NADPH-dependent Disulfide Reductases in the Malaria Parasite

Malaria-associated pathology is caused by the continuous expansion of Plasmodium parasites inside host erythrocytes. To maintain a reducing intracellular milieu in an oxygen-rich environment, malaria parasites have evolved a complex antioxidative network based on two central electron donors, glutathione and thioredoxin. Here, we dissected the in vivo roles of both redox pathways by gene targeting of the respective NADPH-dependent disulfide reductases. We show that Plasmodium berghei glutathione reductase and thioredoxin reductase are dispensable for proliferation of the pathogenic blood stages. Intriguingly, glutathione reductase is vital for extracellular parasite development inside the insect vector, whereas thioredoxin reductase is dispensable during the entire parasite life cycle. Our findings suggest that glutathione reductase is the central player of the parasite redox network, whereas thioredoxin reductase fulfils a specialized and dispensable role for P. berghei. These results also indicate redundant roles of the Plasmodium redox pathways during the pathogenic blood phase and query their suitability as promising drug targets for antimalarial intervention strategies.     

Selenocysteine-containing thioredoxin reductase in C. elegans.

Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases. Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family. The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase. The other gene contains TGA and its product is designated TR-Se. This Sec-containing thioredoxin reductase lacks a canonical Sec insertion sequence element in the 3'-untranslated area of the gene. TR-Se shows greater sequence similarity to mammalian thioredoxin reductase isozymes TR1 and TR2, whereas TR-S is more similar to TR3. TR-Se was identified as a thioredoxin reductase selenoprotein by labeling C. elegans with 75Se and characterizing the resulting 75Se-labeled protein by affinity and other column chromatography and gel-electrophoresis. TR-Se was expressed in Escherichia coli as a selenoprotein when a bacterial SECIS element was introduced downstream of the Sec TGA codon. The data show that TR-Se is the major naturally occurring selenoprotein in C. elegans, and suggest an important role for selenium and the thioredoxin system in this organism.     

The amitochondriate eukaryote Trichomonas vaginalis contains a divergent thioredoxin-linked peroxiredoxin antioxidant system

Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species.     

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.     

EF-hands calcium binding regulates the thioredoxin reductase/thioredoxin electron transfer in human keratinocytes

Thioredoxin reductases purified from Escherichia coli from human metastatic melanoma tissue and from human keratinocytes are subject to allosteric inhibition by calcium. 45Calcium has been used to show that this enzyme contains a single binding site. Bound calcium does not exchange from thioredoxin reductase upon dialysis for 48 hours or upon exposure to 10(-3) M EGTA. An intelligenetics computer analysis yielded a single EF-hands calcium binding site on E. coli thioredoxin reductase with homology to the first EF-hands site on calmodulin. Calcium exchange from the enzyme requires the addition of the natural electron acceptor oxidized thioredoxin which causes a concentration dependent slow exchange. Due to the large conformational change caused by calcium binding to thioredoxin reductase it has been possible to separate Calcium-free and Calcium-bound enzyme by FPLC chromatography. Human keratinocytes contain 5% thioredoxin reductase in their acidic protein cytosol fraction. The influence of extracellular calcium concentration on the intracellular equilibrium between calcium bound versus calcium free thioredoxin reductase has been assessed. This equilibrium was shown to determine the redox status of keratinocytes via the reduction of thioredoxin. Our results provide the first evidence for calcium dependent regulation of redox conditions in the human epidermis.     

Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.

A scheme is described for the large scale purification of thioredoxin, thioredoxin reductase, and glutathione reductase. The scheme is based on an initial separation of thioredoxin from the two reductases by affinity chromatography on agarose-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (agarose-2',5'-ADP). The two reductases were then separated by hydrophobic chromatography and purified separately to homogeneity. Thioredoxin was purified to homogeneity by immunoadsorption to agarose containing immobilized goat anti-thioredoxin. Overall yields for thioredoxin, thioredoxin reductase, and glutathione reductase exceeded 80% in each case. Both reductases exhibit an absorption band at approximately 320 nm which appears due to a residual amount of tightly bound NADP. Presence of this absorption band has no apparent effect on the specific activity of either enzyme.     

Nitrate reductases: Structure,functions, and effect of stress factors

Structural and functional peculiarities of four types of nitrate reductases are considered: assimilatory nitrate reductase of eukaryotes, as well as cytoplasmic assimilatory, membrane-bound respiratory, and periplasmic dissimilatory bacterial nitrate reductases. Arguments are presented showing that eukaryotic organisms are capable of nitrate dissimilation. Data concerning new classes of extremophil nitrate reductases, whose active center does not contain molybdocofactor, are summarized.     

Linked thioredoxin-glutathione systems in platyhelminths

The thioredoxin and glutathione systems play a central role in thiol-disulfide redox homeostasis in many organisms by providing electrons to essential enzymes, and defence against oxidative stress. These systems have recently been characterized in platyhelminth parasites, and the emerging biochemical scenario is the existence of linked processes with the enzyme thioredoxin glutathione reductase supplying reducing equivalents to both pathways. In contrast to their hosts, conventional thioredoxin reductase and glutathione reductase enzymes appear to be absent. Analysis of published data and expressed-sequence tag databases indicates the presence of linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of these parasites.     

Alkyl hydroperoxide reductase from Salmonella typhimurium. Sequence and homology to thioredoxin reductase and other flavoprotein disulfide oxidoreductases

The DNA sequence of the Salmonella typhimurium ahp locus was determined. The locus was found to contain two genes that encode the two proteins (C22 and F52a) that comprise the S. typhimurium alkyl hydroperoxide reductase activity. The predicted sequence of the F52a protein component of the alkyl hydroperoxide reductase was found to be highly homologous to the Escherichia coli thioredoxin reductase protein (34% identity with many conservative substitutions). The homology was found to be particularly striking in the region containing the redox-active cysteines of the thioredoxin reductase molecule, and among the identities were the redox-active cysteines themselves. Aside from the strong similarity to thioredoxin reductase, overall homology between the F52a protein and other flavoprotein disulfide oxidoreductases such as glutathione reductase, dihydrolipoamide dehydrogenase, and mercuric reductase was found to be rather limited, and the conserved active site segment common to the three proteins was not observed within the F52a protein. However, three short segments that have been implicated in FAD and NAD binding were found to be conserved between the F52a protein and the other disulfide reductases. These results suggest that the alkyl hydroperoxide reductase is the second known member of a class of disulfide oxidoreductases which was represented previously by thioredoxin reductase alone; they also allow the putative assignment of several functional domains.     

Glutathionylation of trypanosomal thiol redox proteins

Trypanosomatids, the causative agents of several tropical diseases, lack glutathione reductase and thioredoxin reductase but have a trypanothione reductase instead. The main low molecular weight thiols are trypanothione (N(1),N(8)-bis-(glutathionyl)spermidine) and glutathionyl-spermidine, but the parasites also contain free glutathione. To elucidate whether trypanosomes employ S-thiolation for regulatory or protection purposes, six recombinant parasite thiol redox proteins were studied by ESI-MS and MALDI-TOF-MS for their ability to form mixed disulfides with glutathione or glutathionylspermidine. Trypanosoma brucei mono-Cys-glutaredoxin 1 is specifically thiolated at Cys(181). Thiolation of this residue induced formation of an intramolecular disulfide bridge with the putative active site Cys(104). This contrasts with mono-Cys-glutaredoxins from other sources that have been reported to be glutathionylated at the active site cysteine. Both disulfide forms of the T. brucei protein were reduced by tryparedoxin and trypanothione, whereas glutathione cleaved only the protein disulfide. In the glutathione peroxidase-type tryparedoxin peroxidase III of T. brucei, either Cys(47) or Cys(95) became glutathionylated but not both residues in the same protein molecule. T. brucei thioredoxin contains a third cysteine (Cys(68)) in addition to the redox active dithiol/disulfide. Treatment of the reduced protein with GSSG caused glutathionylation of Cys(68), which did not affect its capacity to catalyze reduction of insulin disulfide. Reduced T. brucei tryparedoxin possesses only the redox active Cys(32)-Cys(35) couple, which upon reaction with GSSG formed a disulfide. Also glyoxalase II and Trypanosoma cruzi trypanothione reductase were not sensitive to thiolation at physiological GSSG concentrations.     

Regeneration of the antioxidant ubiquinol by lipoamide dehydrogenase, thioredoxin reductase and glutathione reductase

Ubiquinol is a powerful antioxidant, which is oxidized in action and needs to be replaced or regenerated to be capable of a sustained effort. This article summarises current knowledge of extramitochondrial reduction of ubiquinone by three flavoenzymes, i.e. lipoamide dehydrogenase, glutathione reductase and thioredoxin reductase, belonging to the same pyridine nucleotide-disulfide oxidoreductase family. These three enzymes are the most efficient extramitochondrial ubiquinone reductases so far described. The reduction of ubiquinone by lipoamide dehydrogenase and glutathione reductase is potently stimulated by zinc and the highest rate of reduction is achieved at acidic pH and the rates are equal with either NADPH or NADH as co-factors. The most efficient ubiquinone reductases are mammalian cytosolic thioredoxin reductases, which are selenoenzymes with a number of biological functions. Reduction of ubiquinone by thioredoxin reductase is in contrast to the other two enzymes investigated, inhibited by zinc and shows a sharp physiological pH optimum at pH 7.5. Furthermore, the reaction is selenium dependent as revealed from experiments using truncated and mutant forms of the enzyme and also in a cellular context by selenium treatment of transfected thioredoxin reductase overexpressing stable cell lines. The reduction of ubiquinone by the three enzymes offers a multifunctional system for extramitochondrial regeneration of an important antioxidant.     

Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.     

The mechanism of action of the nitrosourea anti-tumor drugs on thioredoxin reductase, glutathione reductase and ribonucleotide reductase

The nitrosoureas BCNU, CCNU, ACNU, and Fotemustine covalently deactivate thioredoxin reductase, glutathione reductase and ribonucleotide reductase by alkylating their thiolate active sites. Since thioredoxin reductase and glutathione reductase function as alternative electron donors in the biosynthesis of deoxyribonucleotides, catalyzed by ribonucleotide reductase, the inhibition of these electron transfer systems by the nitrosoureas could determine the cytostatic property of this homologous series of drugs. A detailed study of the kinetics and mechanism for the inhibition of purified thioredoxin reductases from human metastatic melanotic and amelanotic melanomas by the nitrosoureas showed significantly different inhibitor constants. This difference is due to the regulation of these proteins by calcium. Calcium protects thioredoxin reductase from deactivation by the nitrosoureas. In addition, it has been shown that reduced thioredoxin displaces the nitrosourea-inhibitor complex from the active site of thioredoxin reductase to fully reactivate enzyme purified from human metastatic amelanotic melanoma. It has been possible to label the active sites of thioredoxin reductase and glutathione reductase by using chloro[14C]ethyl Fotemustine, resulting in the alkylation of the thiolate active sites to produce chloro[14C]ethyl ether-enzyme inhibitor complexes. These complexes can be reactivated via reduced thioredoxin and reduced glutathione, respectively, by a beta-elimination reaction yielding [14C]ethylene and chloride ions as reaction products.     

Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism

Trypanosomes and leishmania, the causative agents of several tropical diseases, possess a unique redox metabolism which is based on trypanothione. The bis(glutathionyl)spermidine is the central thiol that delivers electrons for the synthesis of DNA precursors, the detoxification of hydroperoxides and other trypanothione-dependent pathways. Many of the reactions are mediated by tryparedoxin, a distant member of the thioredoxin protein family. Trypanothione is kept reduced by the parasite-specific flavoenzyme trypanothione reductase. Since glutathione reductases and thioredoxin reductases are missing, the reaction catalyzed by trypanothione reductase represents the only connection between the NADPH- and the thiol-based redox metabolisms. Thus, cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of trypanothione. Nearly all proteins of the parasite-specific trypanothione metabolism have proved to be essential.