Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

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.     

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.     

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.     

Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense trypanothione reductase.

African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, trypanothione reductase, which acts on a cyclic derivative of glutathione, trypanothione. The high degree of sequence similarity between trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized trypanothione has been determined for trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and functional characterization of thioredoxin from Trypanosoma brucei brucei

Trypanosomes and Leishmania, the causative agents of several tropical diseases, lack the glutathione/glutathione reductase system but have trypanothione/trypanothione reductase instead. The uniqueness of this thiol metabolism and the failure to detect thioredoxin reductases in these parasites have led to the suggestion that these protozoa lack a thioredoxin system. As presented here, this is not the case. A gene encoding thioredoxin has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The single copy gene, which encodes a protein of 107 amino acid residues, is expressed in all developmental stages of the parasite. The deduced protein sequence is 56% identical with a putative thioredoxin revealed by the genome project of Leishmania major. The relationship to other thioredoxins is low. T. brucei thioredoxin is unusual in having a calculated pI value of 8.5. The gene has been overexpressed in Escherichia coli. The recombinant protein is a substrate of human thioredoxin reductase with a K(m) value of 6 microM but is not reduced by trypanothione reductase. T. brucei thioredoxin catalyzes the reduction of insulin by dithioerythritol, and functions as an electron donor for T. brucei ribonucleotide reductase. The parasite protein is the first classical thioredoxin of the order Kinetoplastida characterized so far.     

Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.     

The parasite-specific trypanothione metabolism of trypanosoma and leishmania

The bis(glutathionyl)spermidine trypanothione exclusively occurs in parasitic protozoa of the order Kinetoplastida, such as trypanosomes and leishmania, some of which are the causative agents of several tropical diseases. The dithiol is kept reduced by the flavoenzyme trypanothione reductase and the trypanothione system replaces in these parasites the nearly ubiquitous glutathione/glutathione reductase couple. Trypanothione is a reductant of thioredoxin and tryparedoxin, small dithiol proteins, which in turn deliver reducing equivalents for the synthesis of deoxyribonucleotides as well as for the detoxification of hydroperoxides by different peroxidases. Depending on the individual organism and the developmental state, the parasites also contain significant amounts of glutathione, mono-glutathionylspermidine and ovothiol, whereby all four low molecular mass thiols are directly (trypanothione and mono-glutathionylspermidine) or indirectly (glutathione and ovothiol) maintained in the reduced state by trypanothione reductase. Thus the trypanothione system is central for any thiol regeneration and trypanothione reductase has been shown to be an essential enzyme in these parasites. The absence of this pathway from the mammalian host and the sensitivity of trypanosomatids toward oxidative stress render the enzymes of the trypanothione metabolism attractive target molecules for the rational development of new drugs against African sleeping sickness, Chagas' disease and the different forms of leishmaniasis.     

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.     

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.     

Active site of trypanothione reductase. A target for rational drug design.

The X-ray crystal structure of the enzyme trypanothione reductase, isolated from the trypanosomatid organism Crithidia fasciculata, has been solved by molecular replacement. The search model was the crystal structure of human glutathione reductase that shares approximately 40% sequence identity. The trypanosomal enzyme crystallizes in the tetragonal space group P4(1) with unit cell lengths of a = 128.9 A and c = 92.3 A. The asymmetric unit consists of a homodimer of approximate molecular mass 108 kDa. We present the structural detail of the active site as derived from the crystallographic model obtained at an intermediate stage of the analysis using diffraction data to 2.8 A resolution with an R-factor of 23.2%. This model has root-mean-square deviations from ideal geometry of 0.026 A for bond lengths and 4.7 degrees for bond angles. The trypanosomid enzyme assumes a similar biological function to glutathione reductase and, although similar in topology to human glutathione reductase, has an enlarged active site and a number of amino acid differences, steric and electrostatic, which allows it to process only the unique substrate trypanothione and not glutathione. This protein represents a prime target for chemotherapy of several debilitating tropical diseases caused by protozoan parasites belonging to the genera Trypanosoma and Leishmania. The structural differences between the parasite and host enzymes and their substrates thus provides a rational basis for the design of new drugs active against trypanosomes. In addition, our model explains the results of site-directed mutagenesis experiments, carried out on recombinant trypanothione reductase and glutathione reductases, designed by consideration of the crystal structure of human glutathione reductase.     

Redox control in trypanosomatids, parasitic protozoa with trypanothione-based thiol metabolism

Trypanosomes and leishmania, the causative agents of several tropical diseases, possess a unique redox metabolism which is based on trypanothione. The bis(glutathionyl)spermidine is the central thiol that delivers electrons for the synthesis of DNA precursors, the detoxification of hydroperoxides and other trypanothione-dependent pathways. Many of the reactions are mediated by tryparedoxin, a distant member of the thioredoxin protein family. Trypanothione is kept reduced by the parasite-specific flavoenzyme trypanothione reductase. Since glutathione reductases and thioredoxin reductases are missing, the reaction catalyzed by trypanothione reductase represents the only connection between the NADPH- and the thiol-based redox metabolisms. Thus, cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of trypanothione. Nearly all proteins of the parasite-specific trypanothione metabolism have proved to be essential.     

Peptoid inhibition of trypanothione reductase as a potential antitrypanosomal and antileishmanial drug lead

One route to the design of lead compounds for rational drug design approaches to developing drugs against trypanosomiasis, Chagas' disease and leishmaniasis is to develop novel inhibitors of the parasite-specific enzyme trypanothione reductase. A lead inhibitor based on a peptoid structure was designed in the present study based on the known strong competitive inhibition of trypanothione reductase by N-benzoyl-Leu-Arg-Arg-beta-naphthylamide and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy- beta-naphthylamide. In the target peptoid the arginyl residues were replaced by alkylimidazolium units and the benzyloxycarbonyl group by the benzylaminocarbonyl function. The peptoid was synthesised using t-butoxycarbonyl protection chemistry and couplings were activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The resulting peptoid was shown to be a competitive inhibitor of recombinant trypanothione reductase from Trypanosoma cruzi with a K(i) value of 179 microM and with only weak inhibition of human erythrocyte glutathione reductase (the inhibition of glutathione reductase was at least 291-fold weaker than of trypanothione reductase).     

Ornithine Decarboxylase and Trypanothione Reductase Genes in Leishmania braziliensis guyanensis

. Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC I. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania .     

Ornithine decarboxylase and trypanothione reductase genes in Leishmania braziliensis guyanensis

Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC 1. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania.     

Molecular studies on trypanothione reductase, a target for antiparasitic drugs

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.     

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.     

Thiol metabolism of the trypanosomatids as potential drug targets

Trypanosomatids produce significant amounts of four major low molecular mass thiols, trypanothione, glutathionylspermidine, glutathione, and ovothiol A. Of these, only glutathione is present in cells of the host. All four low molecular mass thiols are directly or indirectly maintained in a reduced state by trypanothione reductase. Available evidence, from gene disruption studies, indicate that this is an essential enzyme. Attempts to exploit trypanothione reductase as a chemotherapeutic target lead to the design of competitive and irreversible inhibitors of the enzyme. A promising route involves the design of redox cyclers interacting specifically with trypanothione reductase as subversive substrates. Progress in studies on the biosynthesis of ovothiol A is summarized.     

Functional and physicochemical characterization of the thioredoxin system in Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses a single thioredoxin that has an unusually high pI value of 8.5 and lacks a conserved aspartyl residue claimed to be involved in catalysis in other thioredoxins. Despite these peculiarities, T. brucei thioredoxin behaves like classical thioredoxins. It is reduced by thioredoxin reductases from different species, serves as donor of reducing equivalents for the ribonucleotide reductase of the parasite, and catalyzes the reduction of protein disulfides. The redox potential of -267 mV was obtained from protein-protein redox equilibration with Escherichia coli thioredoxin. The pK value of T. brucei thioredoxin was determined by two different methods. Carboxamidomethylation of the reduced protein yielded a pK value of 7.4 and generated mono-alkylated protein. The thiolate absorption at 240 nm resulted in a pK of 7.6 and, based on the extinction coefficient of 11.6 mm- 1 cm-1, there are two (or three) cysteines titrating with very similar pK values. A thioredoxin reductase has not yet been detected in any organism of the order Kinetoplastida. T. brucei thioredoxin is spontaneously reduced by trypanothione (bis(glutathionyl)spermidine). Obviously, a specific thioredoxin reductase is not required as thioredoxin reduction can be conducted by the parasite-specific trypanothione/trypanothione reductase system.     

Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design

Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.     

Benzofuranyl 3,5-bis-polyamine derivatives as time-dependent inhibitors of trypanothione reductase

The synthesis and evaluation of 3,5-disubstituted benzofuran derivatives as time-dependent inhibitors of the protozoan oxidoreductase trypanothione reductase are reported. These molecules were designed as simplified mimetics of the naturally occurring spermidine-bridged macrocyclic alkaloid lunarine 1, a known time-dependent inhibitor of trypanothione reductase. In this series of compounds the bis-polyaminoacrylamide derivatives 2-4 were all shown to be competitive inhibitors, but only the bis-4-methyl-piperazin-1-yl-propylacrylamide derivative 4 displayed time-dependent activity. The kinetics of time dependent inactivation of trypanothione reductase by 1 and 4 have been determined and are compared and discussed herein.