Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.12 (trypanothione reductase)
355 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.
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PMID:Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant. 200 14

The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione [N1,N8-bis(glutathionyl)spermidine] by site-directed mutagenesis of two residues. The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore. The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T. congolense trypanothione reductase, and the properties of substrate analogues of trypanothione. Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion.
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PMID:Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase. 205 20

This review focuses on parasite enzymes which are involved in the detoxification of oxygen radicals, and covers the following enzymes: superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase. These enzymes are crucial for parasites to evade oxygen-mediated attack by host leukocytes, both intracellularly and extracellularly. In addition, the newly defined parasite system involving trypanothione, trypanothione peroxidase and trypanothione reductase is discussed.
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PMID:Oxygen detoxifying enzymes in parasites: a review. 248 88

Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate. 1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the kcat/Km ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x 10(6) M-1 s-1) and trypanothione disulfide (4.63 x 10(6) M-1 s-1). 2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 microM) than of trypanothione reductase (IC50 = 200 microM). 3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-5378] a nitrofuran derivative, 2-(5-nitro-2-furanylmethylidene)-N,N'-[1,4-piperazinediylbis (1,3-propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 microM) than for glutathione reductase (IC50 = 10 microM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-1,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor (IC50 = 1 microM) and an NADPH-oxidation-inducing substrate (Km = 14 microM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases.
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PMID:Trypanothione reductase from Trypanosoma cruzi. Catalytic properties of the enzyme and inhibition studies with trypanocidal compounds. 264 89

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.
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PMID:Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase. 316 26

Many parasites--including the causative agents of malaria, Chagas' disease and schistosomiasis--are more susceptible to reactive oxygen species (ROS) than their hosts are. This is manifested by one or more of the following criteria: 1. Susceptibility of the parasite to ROS in vitro; 2. macrophage-based defense mechanisms against the parasite in vivo; 3. successful therapy using agents which lead to oxidative stress; 4. selection advantage (with respect to parasite infections) of human populations whose antioxidant capacity is impaired by a gene defect or by strong oxidants in their staple food. Our laboratory is involved in developing inhibitors against antioxidant enzymes thus mimicking natural experiments. Since glutathione reductase is a protein of known atomic structure the methods of drug design by receptor fit (DDRF) can be applied for this enzyme. Another promising target enzyme is trypanothione reductase which was found so far only in trypanosomatids, and specifically, not in their hosts. Consequently the trypanothione pathway may be a general target in the design of drugs against diseases caused by trypanosomes and leishmanias.
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PMID:Oxidative stress as a defense mechanism against parasitic infections. 350 42

The structural differences between trypanothione reductase of Trypanosoma cruzi and human glutathione reductase, an enzyme of known three-dimensional structure, offer an opportunity for rational drug design against Chagas' disease. As a first step in the analysis of the parasite enzyme we report its purification and characterization. 2.2 mg trypanothione reductase was extracted from 33 g wet weight of cultured epimastigotes or from 4 g lyophilized cells. The flavoenzyme was purified 2400-fold to homogeneity in three steps with an overall yield of 45%. The enzyme is a dimer with a subunit Mr of 50,000. Using NADPH (Km = 5 microM) and trypanothione disulfide (Km = 45 microM) as substrates, a turnover number of 14,200 min-1 was estimated. Trypanothione reductase, the parasite enzyme, and glutathione reductase, the host enzyme, exhibit mutually exclusive specificities for their respective disulfide substrates. When screening cell cultures or column eluates for the presence of trypanothione reductase, a microassay based on Ellman's reagent as indicator was used. A mixture of regioisomeric glutathionylspermidine disulfides isolated from Escherichia coli served as substrate in this microassay. Experimentally, the catalytic cycle of the enzyme can be subdivided into the half-reactions Eox + NADPH + H+----EH2 + NADP+, and EH2 + trypanothione disulfide----Eox + dihydrotrypanothione. This is also true for the crystallized enzyme in the presence of 2 M (NH4)2SO4. The spectral properties of trypanothione reductase both in the oxidized form (Eox) and in the two-electron-reduced form (EH2) closely resemble those of human glutathione reductase. Both proteins contain a flavin and a redox-active disulfide at the catalytic site. After reduction of Eox to EH2, trypanothione reductase can be inactivated by specifically alkylating one of the nascent active-site thiols.
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PMID:Trypanothione reductase from Trypanosoma cruzi. Purification and characterization of the crystalline enzyme. 354 99

The enzyme trypanothione reductase (TR), together with its substrate, the glutathione-spermidine conjugate trypanothione, plays an essential role in protecting parasitic trypanosomatids against oxidative stress and is a target for drug design. Here we show that a naturally occurring spermine derivative, the antihypertensive agent kukoamine A [N1N12-bis(dihydrocaffeoyl)-spermine] inhibits TR as a mixed inhibitor (Ki = 1.8 microM, Kii = 13 microM). Kukoamine shows no significant inhibition of human glutathione reductase (Ki > 10 mM) and thus provides a novel selective drug lead. The corresponding N1N8-bis(dihydrocaffeoyl)spermidine derivative was synthesized and acted as a purely competitive inhibitor with Ki = 7.5 microM. A series of mono- and di-acylated spermines and spermidines were synthesized to gain an insight into the effect of polyamine chain length, the nature and position of the acyl substituent and the importance of conformational mobility. These compounds inhibited TR with Ki values ranging from 11 to 607 microM.
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PMID:Kukoamine A and other hydrophobic acylpolyamines: potent and selective inhibitors of Crithidia fasciculata trypanothione reductase. 748 70

Trypanothione trisulfide was synthesized according to two strategies. It was found to be recognized and reduced by trypanothione reductase as the natural disulfide substrate. At the difference with the mechanism observed for the reduction of glutathione trisulfide by glutathione reductase, the intermediate trypanothione persulfide was rapidly reduced. The enzymatic reduction of another trisulfide derived from an alternative substrate of trypanothione reductase was also studied. The structure of the trisulfide bridge of the substrate (intra- or intermolecular) appeared to be a determining factor in the enzymatic reduction pattern. Moreover, in the case of the alternative substrate of trypanothione reductase, differences of kinetics appeared for the first time between a di- and a trisulfide species. All kinetic parameters are given.
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PMID:Compared recognition of di- and trisulfide substrates by glutathione and trypanothione reductases. 749 72

The flavoprotein thioredoxin reductase catalyzes the reduction of the small redox protein thioredoxin by NADPH. Thioredoxin reductase contains a redox active disulfide and is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes that includes lipoamide dehydrogenase, glutathione reductase, trypanothione reductase, mercuric reductase, and NADH peroxidase. The structure of thioredoxin reductase has recently been determined from X-ray crystallographic data. In this paper, we attempt to correlate the structure with a considerable body of mechanistic data and to arrive at a mechanism consistent with both. The path of reducing equivalents in catalysis by glutathione reductase and lipoamide dehydrogenase is clear. To envisage the path of reducing equivalents in catalysis by thioredoxin reductase, a conformational change is required in which the NADPH domain rotates relative to the FAD domain. The rotation moves the nascent dithiol from its observed position adjacent to the re surface of the flavin ring system toward the protein surface for dithiol-disulfide interchange with the protein substrate thioredoxin and moves the nicotinamide ring of NADPH adjacent to the flavin ring for efficient hydride transfer. Reverse rotation allows reduction of the redox active disulfide by the reduced flavin. This requires that the enzyme pass through a ternary complex; the kinetic evidence for such a complex is discussed.
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PMID:Mechanism and structure of thioredoxin reductase from Escherichia coli. 755 16


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