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The disulfide reducing enzymes glutathione reductase and thioredoxin reductase are highly conserved among bacteria, fungi, worms, and mammals. These proteins maintain intracellular redox homeostasis to protect the organism from oxidative damage. Here we demonstrate the absence of glutathione reductase in Drosophila melanogaster, identify a new type of thioredoxin reductase, and provide evidence that a thioredoxin system supports GSSG reduction. Our data suggest that antioxidant defense in Drosophila, and probably in related insects, differs fundamentally from that in other organisms.

Human thioredoxin reductase is a pyridine nucleotide-disulfide oxidoreductase closely related to glutathione reductase but differing from the latter in having a Cys-SeCys (selenocysteine) sequence as an additional redox center. Because selenoproteins cannot be expressed yet in heterologous systems, we optimized the purification of the protein from placenta with respect to final yield (1-2 mg from one placenta), specific activity (42 units/mg), and selenium content (0.94 ؎ 0.03 mol/mol subunit). The steady state kinetics showed that the enzyme operates by a ping-pong mechanism; the value of k cat was 3330 ؎ 882 min ؊1 , and the K m values were 18 M for NADPH and 25 M for Escherichia coli thioredoxin. The activation energy of the reaction was found to be 53.2 kJ/mol, which allows comparisons of the steady state data with previous pre-steady state measurements. In its physiological, NADPH-reduced form, the enzyme is strongly inhibited by organic gold compounds that are widely used in the treatment of rheumatoid arthritis; for auranofin, the K i was 4 nM when measured in the presence of 50 M thioredoxin. At 1000-fold higher concentrations, that is at micromolar levels, the drugs also inhibited human glutathione reductase and the selenoenzyme glutathione peroxidase. Human thioredoxin reductase (NADPHis a homodimeric flavoenzyme with a subunit size of 55.2 kDa (1-6). This enzyme and other mammalian thioredoxin reductases have recently been shown to be selenoenzymes (2, 7-10). At present, only two other enzyme groups containing selenocysteine are known to occur in mammals, namely glutathione peroxidases and thyroxine deiodinases (EC 3.8.1.4) (8). Because the presence of selenocysteine, so far, does not allow the ectopic production of recombinant TrxR 1 (1, 8), the method for the isolation of the enzyme from human placenta (5) was revisited and improved with respect to speed, yield, and reproducibility. In a previous study (7), we had investigated the reductive half-reaction of the enzyme. In brief, it was shown that the reduction of E ox , the disulfide-containing form of human TrxR, by its substrate NADPH leads to a series of transient enzyme species characterized by charge transfer complexes involving oxidized flavin, reduced flavin, and reoxidized flavin, respectively. The reactions result in a stable TrxR species containing reoxidized flavin, the active site pair Cys-57/Cys-62 as a dithiol, and an additional reduced redox active group, probably the Cys-495/SeCys-496 center. The nascent thiolate of Cys-62 forms a charge transfer complex with the flavin, which has a typical absorbance at 540 nm. Thus, human thioredoxin reductase mechanistically resembles glutathione reductase and is distinct from bacterial TrxR (7,11,12). Employing steady state kinetics, we have now continued investigating the catalytic mechanism of human thioredoxin reductase.Studies with the gold compound aurothioglucose on human glutathione peroxidase (13) and human iodothyronine deiodinase type 1 (14), as well as preliminary studies on thioredoxi...

Thioredoxin reductase, lipoamide dehydrogenase, and glutathione reductase are members of the pyridine nucleotide-disulfide oxidoreductase family of dimeric f lavoenzymes. The mechanisms and structures of lipoamide dehydrogenase and glutathione reductase are alike irrespective of the source (subunit M r Ϸ55,000). Although the mechanism and structure of thioredoxin reductase from Escherichia coli are distinct (M r Ϸ35,000), this enzyme must be placed in the same family because there are significant amino acid sequence similarities with the other two enzymes, the presence of a redox-active disulfide, and the substrate specificities. Thioredoxin reductase from higher eukaryotes on the other hand has a M r of Ϸ55,000 Nitrosoureas of the carmustine type inhibit only the NADPH reduced form of human thioredoxin reductase. These compounds are widely used as cytostatic agents, so this enzyme should be studied as a target in cancer chemotherapy. In conclusion, three lines of evidence indicate that the mechanism of human thioredoxin reductase is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase.

Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H 2 O 2 -based oxidative stress.Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal K i for the stoichiometric inhibition being 4 nm. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjo Ègren's syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance.TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria.Keywords: antioxidant systems; aurothioglucose; carmustine; diselenide; drug resistance; Epstein±Barr virus; leprosy; malaria; rheumatoid arthritis; selenium metabolism.Thioredoxin reductase (TrxR; EC 1.6.4.5; thioredoxin-S 2 1 NADPH 1 H 1 O thioredoxin-(SH) 2 1 NADP 1 ) belongs to a family of glutathione reductase-like homodimeric flavoenzymes [1]. Genetic and mechanistic aspects of TrxRs from different species are covered in more detail by other articles in this review series and elsewhere [1±5]. As shown in Fig. 1, the 35-kDa (subunit M r ) TrxRs occurring in prokaryotes but also in plants and fungi differ fundamentally from the 55-to 60-kDa TxRs that have been identified so far in mammals, Caenorhabditis elegans, Drosophila melanogaster, and in the malaria parasite Plasmodium falciparum. The high M r TrxRs contain a C-terminal peripheral redox centre that communicates with the central redox-active catalytic site [6]. Whereas the peripheral redox centre of P. falciparum TrxR is represented by Cys535 and Cys540 [7] and by Cys489±Cys490 in D. melanogaster (S. M. Kanzok, H. Bauer, R. H. Schirmer & K. Becker, unpublished results), all known mammalian TrxRs possess ...

In most living cells, redox homeostasis is based both on the glutathione and the thioredoxin system. In the malaria parasite Plasmodium falciparum antioxidative proteins represent promising targets for the development of antiparasitic drugs. We cloned and expressed a thioredoxin of P. falciparum (pftrx), and we improved the stable expression of the thioredoxin reductase (PfTrxR) of the parasite by multiple silent mutagenesis. Both proteins were biochemically characterized and compared with the human host thioredoxin system. Intriguingly, the 13-kDa protein PfTrx is a better substrate for human TrxR (K m ‫؍‬ 2 M, k cat ‫؍‬ 3300 min ؊1 ) than for P. falciparum TrxR (K m ‫؍‬ 10.4 M, k cat ‫؍‬ 3100 min ؊1 ). Possessing a midpoint potential of ؊270 mV, PfTrx was found to reduce the disease-related metabolites S-nitrosoglutathione and GSSG. The rate constant k 2 for the reaction between reduced P. falciparum thioredoxin and GSSG was determined to be 0.039 M ؊1 min ؊1 at 25°C and pH 7.4. The k 2 for thioredoxins from man, Drosophila melanogaster, and Escherichia coli was ϳ5 times lower. Our data suggest that GSSG reduction can be supported at a high rate by the TrxR/Trx system in glutathione reductase-deficient cells; this may be relevant for certain stages of the malarial parasite but also for cells containing high [GSSG] of other organisms like dormant forms of Neurospora, glutathione reductasedeficient yeast mutants, or CD4 ؉ lymphocytes of AIDS patients.

Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage.

Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -SerCys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.