Recombinant putative glutathione reductase of Plasmodium falciparum exhibits thioredoxin reductase activity

Müller, Sylke; Gilberger, Tim‐Wolf; Pm, Färber; Becker, Katja; Rh, Schirmer; Rd, Walter

doi:10.1016/0166-6851(96)02694-1

Cited by 56 publications

(31 citation statements)

References 15 publications

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“…With respect to an essential role of SeCys, it is worth mentioning that high M r TrxR of the malaria parasite Plasmodium falciparum has a penultimate CysXXXXCys motif. According to the deduced amino acid sequence, the enzyme does not contain SeCys, however (12,46).…”

Section: Discussionmentioning

confidence: 99%

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The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Arscott

Gromer

Schirmer

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Discussionmentioning

confidence: 99%

“…Moreover, complete DNA sequences have been published for TrxR from humans (1,11) and Plasmodium falciparum (ref. 12 and references therein). The deduced amino acid sequences of these high M r TrxR are much more closely related to the sequences of GR from humans (35% identity) or E. coli (40% identity) than to the sequence of eTrxR (24% identity) (1,13).…”

mentioning

confidence: 99%

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Arscott

Gromer

Schirmer

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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224

235

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“…The gene was identified in the genome of P. falciparum, cloned, and expressed as a recombinant functional flavoenzyme. [152,[182][183][184] Subsequently, Müller and co-workers used gene-ablation and gene-substitution techniques to show that thioredoxin reductase is an essential enzyme for the blood stages of P. falciparum. [153,154] These studies validated thioredoxin reductase as a drug target.…”

Section: Thioredoxin Reductase As a Validated Drug Targetmentioning

confidence: 99%

Dithiol Proteins as Guardians of the Intracellular Redox Milieu in Parasites: Old and New Drug Targets in Trypanosomes and Malaria‐Causing Plasmodia

2005

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Parasitic diseases such as sleeping sickness, Chagas' heart disease, and malaria are major health problems in poverty-stricken areas. Antiparasitic drugs that are not only active but also affordable and readily available are urgently required. One approach to finding new drugs and rediscovering old ones is based on enzyme inhibitors that paralyze antioxidant systems in the pathogens. These antioxidant ensembles are essential to the parasites as they are attacked in the human host by strong oxidants such as peroxynitrite, hypochlorite, and H2O2. The pathogen-protecting system consists of some 20 thiol and dithiol proteins, which buffer the intraparasitic redox milieu at a potential of -250 mV. In trypanosomes and leishmania the network is centered around the unique dithiol trypanothione (N1,N8-bis(glutathionyl)spermidine). In contrast, malaria parasites have a more conservative dual antioxidative system based on glutathione and thioredoxin. Inhibitors of antioxidant enzymes such as trypanothione reductase are, indeed, parasiticidal but they can also delay or prevent resistance against a number of other antiparasitic drugs.

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“…The enzymes GR (23), TrxR (29,42), and mitochondrial LipDH of P. falciparum (38) were expressed in recombinant forms, purified, and assayed at 25°C as described previously. For the mammalian counterparts, we used recombinant human GR (44), human TrxR (from placenta as well as the recombinant enzyme) (24,27), and pig heart mitochondrial LipDH (obtained from Sigma), the best-studied mammalian LipDH (4,56).…”

Section: Methodsmentioning

confidence: 99%

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

Buchholz

Schirmer

Eubel

et al. 2008

Antimicrob Agents Chemother

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Methylene blue (MB) has experienced a renaissance mainly as a component of drug combinations against Plasmodium falciparum malaria. Here, we report biochemically relevant pharmacological data on MB such as rate constants for the uncatalyzed reaction of MB at pH 7.4 with cellular reductants like NAD(P)H (k ‫؍‬ 4 M ؊1 s ؊1 ), thioredoxins (k ‫؍‬ 8.5 to 26 M ؊1 s ؊1 ), dihydrolipoamide (k ‫؍‬ 53 M ؊1 s ؊1 ), and slowly reacting glutathione. As the disulfide reductases are prominent targets of MB, optical tests for enzymes reducing MB at the expense of NAD(P)H under aerobic conditions were developed. The product leucomethylene blue (leucoMB) is auto-oxidized back to MB at pH 7 but can be stabilized by enzymes at pH 5.0, which makes this colorless compound an interesting drug candidate. MB was found to be an inhibitor and/or a redox-cycling substrate of mammalian and P. falciparum disulfide reductases, with the k cat values ranging from 0.03 s ؊1 to 10 s ؊1 at 25°C. Kinetic spectroscopy of mutagenized glutathione reductase indicates that MB reduction is conducted by enzyme-bound reduced flavin rather than by the active-site dithiol Cys 58 /Cys 63 . The enzyme-catalyzed reduction of MB and subsequent auto-oxidation of the product leucoMB mean that MB is a redox-cycling agent which produces H 2 O 2 at the expense of O 2 and of NAD(P)H in each cycle, turning the antioxidant disulfide reductases into pro-oxidant enzymes. This explains the terms subversive substrate or turncoat inhibitor for MB. The results are discussed in cellpathological and clinical contexts. Methylene blue (MB or MBϩ ) [also known as methylthionine hydrochloride or 3,7-bis(dimethylamino)phenothiazin-5-ium chloride] was the very first synthetic compound to be used as a drug. Paul Ehrlich, who introduced the concept of modern target-based chemotherapy using MB as an example, and Paul Guttmann described the compound as being an effective antimalarial agent (28). Despite its beneficial antimalarial activity, the drug disappeared from the scene because up-and-coming compounds such as chloroquine were more effective; in addition, soldiers resented taking MB because of inevitable but harmless side effects: green or blue urine and bluish sclerae (47, 55).After MB was revisited as an antimalarial agent (6, 53) and found to be an inhibitor of Plasmodium falciparum glutathione (GSH) reductase (GR) (23), it was studied as a partner drug in antimalarial drug combinations (2,39,48,51).Compared with other antiparasitic agents, MB is affordable and registered in most countries (as the treatment of choice for acute and chronic methemoglobinemia [16,17,36]), and it can be made internationally available in sufficient dosages (51). The price for treating a malaria episode in a child with an MB-containing drug combination would be less than €0.50 (40). Drug resistance has not been reported for MB and could not be provoked in rodent malaria models (52, 53).Because of its favorable properties, including the differential staining of cell biological structures and protein cr...

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Recombinant putative glutathione reductase of Plasmodium falciparum exhibits thioredoxin reductase activity

Cited by 56 publications

References 15 publications

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli

Dithiol Proteins as Guardians of the Intracellular Redox Milieu in Parasites: Old and New Drug Targets in Trypanosomes and Malaria‐Causing Plasmodia

Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues fromPlasmodium falciparum

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