Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.
Mol Microbiol 1992 Nov
PMID:Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism. 145 51

Trypanothione reductase (TR) is a target for drug design since it is unique to trypanosomatids, substituting for the otherwise ubiquitous enzyme, glutathione reductase. We report the cloning and sequencing of several cDNAs and genes encoding Crithidia fasciculata TR, the structure of which has recently been solved by crystallography. Single base polymorphisms are detected in cDNAs (containing 80% of the coding sequence) and two different genomic clones, including a glutamine to glutamate change in the C-terminal region of the TR coding region; other nucleotide changes are silent. Homology (from genomic clones, both of which contained signals appropriate for expression) to the Trypanosoma congolense gene was 63% at the nucleic acid level, with 68% amino acid identity; the significance of homologies to human and Escherichia coli glutathione reductase sequences is discussed. Polymorphic sites in the genomic clones included sites found in the cDNAs, indicating that differences existing in the genomic sequence are real, and propagated to RNA.
Mol Biochem Parasitol 1992 Jan
PMID:Cloning, sequencing, and demonstration of polymorphism in trypanothione reductase from Crithidia fasciculata. 154 16

Trypanothione reductase, a flavoprotein disulfide reductase specific to trypanosomatid parasites, has been crystallized by vapor diffusion of a protein solution (10 mg/ml) against 22% polyethylene glycol (average Mr 8000) containing 100 mM-ammonium sulfate. Crystals of a size suitable for structure determination by X-ray diffraction have been obtained by seeding protein solutions with smaller crystals. The space-group is P21 (a = 60.9 A, b = 161.8 A, c = 58.4 A, beta = 99.1 degrees). The molecular mass and volume of the unit cell suggest that there is a dimer of the enzyme in the asymmetric unit, and this is confirmed by self-rotation functions calculated using data to 4.5 A resolution. The crystals diffract to beyond 3 A resolution. Crystals of another P21 form (a = 91.3 A, b = 114.4 A, c = 92.0 A, beta = 141.3 degrees) are observed to grow under similar conditions.
J Mol Biol 1990 Oct 05
PMID:Preliminary crystallographic analysis of trypanothione reductase from Crithidia fasciculata. 223 7

The effect of D,L-alpha-difluoromethylornithine (DFMO) on thiol and polyamine levels in Trypanosoma brucei was investigated by isolating trypanosomes from infected rats treated with DFMO for 12-48 h. Concentrations of thiols, polyamines and other amino-compounds were measured by an automated high-performance liquid chromatography method. The levels of DFMO in rat plasma (0.02-1.34 mM) is similar to that found in the parasites (0.27-0.99 mM), concentrations which exceed the Ki of DFMO for T. brucei ornithine decarboxylase. Treatment with DFMO increases intracellular levels of ornithine, S-adenosylmethionine and decarboxylated S-adenosylmethionine and decreases putrescine and spermidine. Putrescine is undetectable after 12 h treatment with DFMO and after 48 h spermidine is decreased by 76%. By 48 h, the spermidine-glutathione conjugates glutathionylspermidine and dihydrotrypanothione (bis(glutathionyl)spermidine) are also decreased by 41 and 66%, respectively. In contrast, levels of glutathione show a slight increase. These changes in metabolite levels are consistent with the biosynthetic pathway proposed for Crithidia fasciculata, where trypanothione is synthesized from spermidine and glutathione via the intermediates N1- and N8-glutathionyl-spermidine. Trypanothione is thought to have two important roles in trypanosomatid metabolism: the maintenance of intracellular thiols in the correct redox state and in the removal of hydrogen peroxide and other hydroperoxides. Thus, it is proposed that depletion of this metabolite may be an important contributory factor to the selective toxic effect of DFMO, particularly in its synergistic effect with other trypanocidal drugs.
Mol Biochem Parasitol 1987 Jun
PMID:In vivo effects of difluoromethylornithine on trypanothione and polyamine levels in bloodstream forms of Trypanosoma brucei. 311 34

A trypanothione reductase activity is present in all the main differentiation stages of Trypanosoma cruzi, amastigotes having the highest activity, and trypomastigotes the lowest. Trypanothione reductase could not be induced in epimastigotes exposed to H2O2. The trypanocidal drug crystal violet was a potent inhibitor of T. cruzi trypanothione reductase in vitro. The inhibition was competitive with respect to trypanothione with a Ki of 5.3 +/- 0.5 microM, uncompetitive with NADPH, and increased below pH 7.0 and above pH 8.0. Crystal violet, however, was not able to decrease the level of total reduced thiols in intact cells. Dihydrotrypanothione but not reduced glutathione, protected the enzyme from inhibition by crystal violet.
Mol Biochem Parasitol 1994 Oct
PMID:Inhibition of Trypanosoma cruzi trypanothione reductase by crystal violet. 787 Jan 35

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in the trypanosomatids. We report here the cloning by expression of the Leishmania donovani gene. It is single copy, expresses a 2.6-kb transcript and a 52-kDa protein and is located on a 1.1-Mbp chromosome. The 491 amino acid sequence has 76% similarity to Crithidia fasciculata and 67% similarity to Trypanosoma cruzi, Trypanosoma congolense and Trypanosoma brucei TR. Residues recognising the adenosine pyrophosphate moiety of NADPH and FAD, and residues in the catalytic site segment (A47-A67) involving electron transfer from TR to trypanothione disulphide (T(S)2) were completely conserved. Thus inhibitors of TR are likely to be active against the enzyme from all the parasitic trypanosomatids. Two peptide inserts (39-47, 131-140) seen in other TR genes and a C-terminal extension of 19 residues were also present. When the gene was introduced back into L. donovani at high copy number using the pTEX expression vector, we detected elevated expression of TR RNA and a 14-fold increase in TR activity. Transfection and overexpression of the TR gene will facilitate studies of gene function and of the dependence of trypanosomatids on TR for protection against oxidative stress.
Mol Biochem Parasitol 1994 Apr
PMID:The structure, organization, and expression of the Leishmania donovani gene encoding trypanothione reductase. 793 7

Trypanothione, a metabolite specific to trypanosomatid parasites, is enzymatically synthesized from spermidine and glutathione by the consecutive action of the ATP-dependent carbon-nitrogen ligases, glutathionylspermidine synthetase and trypanothione synthetase. As part of our programme aimed at developing inhibitors of these enzymes, we have synthesized a series of analogues of glutathione (gamma-L-Glu-L-Cys-Gly) and tested them as substrates or inhibitors of glutathionylspermidine synthetase. Recognition at the gamma-glutamyl moiety appears to be essential, as any modification of this part of glutathione results in a total loss of activity as a substrate. Alkylation of the thiol side chain of cysteine with methyl, ethyl or propyl groups yields analogues with catalytic efficiencies (kcat/Km) as substrates equivalent to or better than glutathione. In contrast, the bulkier S-butyl analogue was a much poorer substrate. Substitution of L-Cys by amino acids with an alkyl side-chain is also well tolerated giving relative catalytic efficiencies of 1.1 and 1.5 for peptide analogues containing L-Val and L-Ile respectively. Other analogues, where the bulk of the alkyl chain is increased further (as in L-Leu or L-Phe) or where the glycine moiety is replaced with L-Ala, are inhibitors rather than substrates.
Mol Biochem Parasitol 1997 Jan
PMID:Characterization of the peptide substrate specificity of glutathionylspermidine synthetase from Crithidia fasciculata. 904 18

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use.
Mol Microbiol 1998 Jul
PMID:Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus. 972 Aug 80

Leishmania is a protozoan parasite responsible for significant morbidity and mortality worldwide. Few parasites have been subjected to proteomic analysis to date, but a genome sequencing project for Leishmania major is currently underway, making these studies possible. Here we present a high resolution proteome for L. major comprising almost 3700 spots, making it the most complete two-dimensional gel representation of a parasite proteome generated to date. We have identified a number of landmark proteins by mass spectrometry and show that several of these are valid for the related species Leishmania donovani infantum. We have also observed several forms and fragments of alpha- and beta-tubulins and show that the number and amount of these fragments increase with the age of the parasite culture. Trypanothione reductase (TRYR), which replaces glutathione reductase in trypanosomatid parasites, is an essential protein specific to these parasites and as such is under considerable scrutiny as a drug target. Two-dimensional gel analysis of a L. major strain overexpressing TRYR revealed increased amounts of five spots, all at the predicted molecular weight for TRYR and differing by 0.08 pH units in pI. Mass spectrometry identified four of these as TRYR, leading to the novel suggestion that it could be post-translationally modified. Finally quantitative comparative analysis of a methotrexate-resistant mutant of L. major generated in vitro found that a known primary resistance mediator, the pteridine reductase PTR1, was overexpressed. This constitutes the first proteomic analysis of drug resistance in a parasite and also the clearest identification of a primary drug resistance mechanism using this approach. Together these results provide a framework for further proteomic studies of Leishmania species and demonstrate that these tools are valuable for the essential study of potential drug targets and drug resistance mechanisms.
Mol Cell Proteomics 2003 Mar
PMID:Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms. 1264 73

Trypanothione [N(1),N(8)-bis(glutathionyl)spermidine] plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids. In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity. To determine which route operates in T. brucei, we have cloned and expressed a single copy gene with similarity to C. fasciculata and T. cruzi TRYS. The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH. The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)). At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively. Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T. cruzi. The enzyme has amidase activity that can be inhibited by iodoacetamide. Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain. Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C. fasciculata.
Mol Biochem Parasitol 2003 Sep
PMID:Properties of trypanothione synthetase from Trypanosoma brucei. 1296 9


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