Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata

The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1,N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 microM and 379 microM, respectively. Since the disulfide form of N1-monoglutathionylspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

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.     

Bis(glutathionyl)spermine and other novel trypanothione analogues in Trypanosoma cruzi

Trypanosomatids differ from other cells in their ability to conjugate glutathione with the polyamine spermidine to form the antioxidant metabolite trypanothione (N1,N8-bis(glutathionyl)spermidine). In Trypanosoma cruzi, trypanothione is synthesized by an unusual trypanothione synthetase/amidase (TcTryS) that forms both glutathionylspermidine and trypanothione. Because T. cruzi is unable to synthesize putrescine and is dependent on uptake of exogenous polyamines by high affinity transporters, synthesis of trypanothione may be circumstantially limited by lack of spermidine. Here, we show that the parasite is able to circumvent the potential shortage of spermidine by conjugating glutathione with other physiological polyamine substrates from exogenous sources (spermine, N8-acetylspermidine, and N-acetylspermine). Novel thiols were purified from epimastigotes, and structures were determined by matrix-assisted laser desorption ionization time-of-flight analysis to be N1,N12-bis(glutathionyl)spermine, N1-glutathionyl-N8-acetylspermidine, and N1-glutathionyl-N12-acetylspermine, respectively. Structures were confirmed by enzymatic synthesis with recombinant TcTryS, which catalyzes formation of these compounds with kinetic parameters equivalent to or better than those of spermidine. Despite containing similar amounts of spermine and spermidine, the epimastigotes, trypomastigotes, and amastigotes of T. cruzi preferentially synthesized trypanothione. Bis(glutathionyl)spermine disulfide is a physiological substrate of recombinant trypanothione reductase, comparable to trypanothione and homotrypanothione disulfides. The broad substrate specificity of TcTryS could be exploited in the design of polyamine-based inhibitors of trypanothione metabolism.     

Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase

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.     

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.     

Trypanothione reductase from Trypanosoma cruzi. Catalytic properties of the enzyme and inhibition studies with trypanocidal compounds

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.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

Synthesis of asymmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 2

Summary The synthesis of asymmetrical disulfides, based on Zervas' inter-mediate, monocarbobenzoxy-L-cystine, has been developed. A series of substrate analogues of trypanothione disulfide (TSST) and glutathione disulfide (GSSG) are described, where the spermidine ring of (TSST) has been replaced by 3-dimethylaminopropylamine (DMAPA). The free amino group in Zervas' product was condensed with phenylalanyl, tryptophanyl or glutamyl residues, while the carbobenzoxy group was unaffected under the reaction conditions employed. The same synthetic approach was applied in the design of analogues of glutathione disulfide (GSSG).     

Preparative enzymatic synthesis of trypanothione and trypanothione analogues

Trypanosomatids, the causative agents of several tropical diseases, have a unique thiol metabolism based on trypanothione [bis(glutathionyl)spermidine]. Enzymes of the pathway are attractive drug target molecules but the availability of trypanothione remains an obstacle. Here, we present a convenient method for the production of trypanothione and trypanothione disulfide in >200 mg quantities using a mutant of Crithidia fasciculata trypanothione synthetase in which Cys59 has been replaced by an alanine residue. The reagent costs less than 1% of the commercial price of trypanothione disulfide. The protocol also allows the synthesis of related glutathione conjugates. It will greatly facilitate the thorough analysis of this parasite’s metabolism and drug screening approaches against trypanothione-dependent enzymes.     

Synthesis of symmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 1

Summary The synthesis of a series of symmetrical disulfides as potential substrates of trypanothione reductase and glutathione reductase was described. The key intermediate in the synthetic approach was the choice of S-^tbutylmercapto-L-cysteine (1). The spermidine ring in the native substrate, trypanothione disulfide (TSST), was replaced with 3-dimethyl-aminopropylamine (DMAPA), while the-Glu moiety was replaced by phenylalanyl or tryptophanyl residues. The same modifications in the-Glu moiety of glutathione disulfide (GSSG) were applied.     

A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi

Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione [N(1),N(8)-bis(glutathionyl)spermidine], a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions. In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively. Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS. Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS. When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione [N(1),N(9)-bis(glutathionyl)aminopropylcadaverine], metabolites that have been previously identified in T. cruzi, but not in C. fasciculata. Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate. The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine.     

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.     

Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase.

Mycothione reductase from the human pathogen Mycobacterium tuberculosis has been cloned, expressed in Mycobacterium smegmatis, and purified 145-fold to homogeneity in 43% yield. Amino acid sequence alignment of mycothione reductase with the functionally homologous glutathione and trypanothione reductase indicates conservation of the catalytically important redox-active disulfide, histidine-glutamate ion pair, and regions involved in binding both the FAD cofactor and the substrate NADPH. The homogeneous 50 kDa subunit enzyme exists as a homodimer and is NADPH-dependent and highly specific for the structurally unique low-molecular mass disulfide, mycothione, exhibiting Michaelis constants of 8 and 73 microM for NADPH and mycothione, respectively. HPLC analysis indicated the presence of 1 mol of bound FAD per monomer as the cofactor exhibiting an absorption spectrum with a lambda(max) at 462 nm with an extinction coefficient of 11 300 M(-)(1) cm(-)(1). The reductive titration of the enzyme with NADH indicates the presence of a charge-transfer complex of one of the presumptive catalytic thiolates and FAD absorbing at ca. 530 nm. Reaction with serially truncated mycothione and other disulfides and pyridine nucleotide analogues indicates a strict minimal disulfide substrate requirement for the glucosamine moiety of mycothione. The enzyme exhibits bi-bi ping-pong kinetics with both disulfide and quinone substrates. Transhydrogenase activity is observed using NADH and thio-NADP(+), confirming the kinetic mechanism. We suggest mycothione reductase as the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.     

Trypanothione synthesis in crithidia revisited

In Crithidia fasciculata the biosynthesis of trypanothione (N(1),N(8)-bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBanktrade mark accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg(2+)-ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K(m) values for GSH, Mg(2+)-ATP, and glutathionylspermidine were 407, 222, and 480 microm, respectively, and the k(cat) was 8.7 s(-1) for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.     

Dual action of antimonial drugs on thiol redox metabolism in the human pathogen Leishmania donovani

Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (Sb(III)) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, Sb(III) decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, Sb(III) inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.     

Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites

A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.     

Glyoxalase II of African trypanosomes is trypanothione-dependent

The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.     

The trypanothione-thiol system in Trypanosoma cruzi as a key antioxidant mechanism against peroxynitrite-mediated cytotoxicity

Peroxynitrite, the reaction product between superoxide (O(*2)) and nitric oxide (*NO), is a powerful oxidizing species that contributes to macrophage competence against pathogens. In this context, peroxynitrite appears to play an important role in controlling infection by Trypanosoma cruzi, the unicellular parasite responsible for Chagas disease. T. cruzi contains various enzyme systems for the decomposition of hydroperoxides, all of which involve the participation of the low-molecular-weight dithiol trypanothione (N(1),N(8)-bis(glutathionyl)spermidine) as a critical redox partner. A large fraction of the trypanothione-dependent antioxidant capacity of T. cruzi is linked to the tryparedoxin-tryparedoxin peroxidase system which has critical protein thiol groups. In this report we demonstrate that dihydrotrypanothione is readily consumed during peroxynitrite challenge to cells to yield the corresponding trypanothione disulfide. On the other hand, glutathione, which is present in T. cruzi at lower concentrations than trypanothione, is consumed to a much lesser extent and mainly evolves to glutathione-protein mixed disulfides. The inhibition of glutathione biosynthesis by buthionine sulfoximine, which decreases glutathione concentration to 10% of control after 20 h, neither affects the concentration of dihydrotrypanothione nor sensitizes T. cruzi to peroxynitrite-mediated cytotoxicity. On the other hand, pretreatment of T. cruzi with diamide, which leads to a significant depletion (>70%) of dihydrotrypanothione, largely increases the extent of cellular nitration and inhibition of cell growth caused by peroxynitrite. Altogether, our findings support a key protective role for dihydrotrypanothione and the trypanothione-dependent antioxidant system in T. cruzi against peroxynitrite, which may facilitate the survival of trypanosomes within the oxidative environment of activated macrophages.     

Trypanothione-dependent synthesis of deoxyribonucleotides by Trypanosoma brucei ribonucleotide reductase

Trypanosoma brucei, the causative agent of African sleeping sickness, synthesizes deoxyribonucleotides via a classical eukaryotic class I ribonucleotide reductase. The unique thiol metabolism of trypanosomatids in which the nearly ubiquitous glutathione reductase is replaced by a trypanothione reductase prompted us to study the nature of thiols providing reducing equivalents for the parasite synthesis of DNA precursors. Here we show that the dithiol trypanothione (bis(glutathionyl)spermidine), in contrast to glutathione, is a direct reductant of T. brucei ribonucleotide reductase with a K(m) value of 2 mm. This is the first example of a natural low molecular mass thiol directly delivering reducing equivalents for ribonucleotide reduction. At submillimolar concentrations, the reaction is strongly accelerated by tryparedoxin, a 16-kDa parasite protein with a WCPPC active site motif. The K(m) value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin is about 4 micrometer. The disulfide form of trypanothione is a powerful inhibitor of the tryparedoxin-mediated reaction that may represent a physiological regulation of deoxyribonucleotide synthesis by the redox state of the cell. The trypanothione/tryparedoxin system is a new system providing electrons for a class I ribonucleotide reductase, in addition to the well known thioredoxin and glutaredoxin systems described in other organisms.     

New enzymes for old: redesigning the coenzyme and substrate specificities of glutathione reductase.

A set of amino acid side chains that confer specificity for the coenzyme NADPH and the substrate glutathione in the flavoprotein disulphide oxidoreductase, glutathione reductase, has been identified. Systematic replacement of these amino acid residues in the coenzyme-binding site switches the specificity of the enzyme from its natural strong preference for NADPH to a marked preference for NADH. The amino acids replaced all lie in a structural motif within the dinucleotide-binding domain of the protein. Since this domain is a feature common to most dehydrogenases (reductases) that use nicotinamide coenzymes, it may be that the coenzyme specificities of all such enzymes can be manipulated in this way. Similarly, amino acid residues involved in the selective recognition of trypanothione by trypanothione reductase, an enzyme related to glutathione reductase and exclusive to trypanosomatids, were identified. Suitable mutation of the corresponding residues in E. coli glutathione reductase switched its substrate specificity towards trypanothione. A better understanding of the substrate specificity of these enzymes could open up a route to the chemotherapy of trypanosomal infections.