Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.     

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (?NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ?NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.     

Platyhelminth mitochondrial and cytosolic redox homeostasis is controlled by a single thioredoxin glutathione reductase and dependent on selenium and glutathione

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The K(m) and k(cat) values for mitochondrial and cytosolic thioredoxins (9.5 microm and 131 s(-1), 34 microm and 197 s(-1), respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 microm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.     

Linked thioredoxin-glutathione systems in platyhelminth parasites: alternative pathways for glutathione reduction and deglutathionylation

In most organisms, thioredoxin (Trx) and/or glutathione (GSH) systems are essential for redox homeostasis and deoxyribonucleotide synthesis. Platyhelminth parasites have a unique and simplified thiol-based redox system, in which the selenoprotein thioredoxin-glutathione reductase (TGR), a fusion of a glutaredoxin (Grx) domain to canonical thioredoxin reductase domains, is the sole enzyme supplying electrons to oxidized glutathione (GSSG) and Trx. This enzyme has recently been validated as a key drug target for flatworm infections. In this study, we show that TGR possesses GSH-independent deglutathionylase activity on a glutathionylated peptide. Furthermore, we demonstrate that deglutathionylation and GSSG reduction are mediated by the Grx domain by a monothiolic mechanism and that the glutathionylated TGR intermediate is resolved by selenocysteine. Deglutathionylation and GSSG reduction via Grx domain, but not Trx reduction, are inhibited at high [GSSG]/[GSH] ratios. We found that Trxs (cytosolic and mitochondrial) provide alternative pathways for deglutathionylation and GSSG reduction. These pathways are operative at high [GSSG]/[GSH] and function in a complementary manner to the Grx domain-dependent one. Despite the existence of alternative pathways, the thioredoxin reductase domains of TGR are an obligate electron route for both the Grx domain- and the Trx-dependent pathways. Overall, our results provide an explanation for the unique array of thiol-dependent redox pathways present in parasitic platyhelminths. Finally, we found that TGR is inhibited by 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7), giving further evidence for NO donation as a mechanism of action for oxadiazole N-oxide TGR inhibitors. Thus, NO donors aimed at TGR could disrupt the entire redox homeostasis of parasitic flatworms.     

Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni

Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the lags. A Sec-597 variant was still able to reduce a variety of substrates, albeit slowly, showing that selenocysteine is important but is not the sole determinant for the broad substrate tolerance of the enzyme. Our data show that Cys-520 and Cys-574 are not likely to be involved in the catalytic mechanism.     

A recombinant thioredoxin-glutathione reductase from Fasciola hepatica induces a protective response in rabbits

Antioxidant systems are fundamental components of host–parasite interactions, and often play a key role in parasite survival. Here, we report the cloning, heterologous expression, and characterization of a thioredoxin glutathione reductase (TGR) from Fasciola hepatica. The deduced polypeptide sequence of the cloned open reading frame (ORF) confirmed the experimental N-terminus previously determined for a native F. hepatica TGR showing thioredoxin reductase (TR) activity. The sequence revealed the presence of a fusion between a glutaredoxin (Grx) and a TR domain, similar to that previously reported in Schistosoma mansoni and Echinococcus granulosus. The F. hepatica TGR sequence included an additional redox active center (ACUG; U being selenocysteine) located at the C-terminus. The addition of a recombinant selenocysteine insertion sequence (SECIS) element in the Escherichia coli expression vector, or the substitution of the native selenocysteine by a cysteine, indicated the relevance of this unusual amino acid residue for the activity of F. hepatica TGR. Rabbit vaccination with recombinant F. hepatica TGR reduced the worm burden by 96.7% following experimental infection, further supporting the relevance of TGR as a promising target for anti Fasciola treatments.     

Mammalian selenoprotein thioredoxin-glutathione reductase. Roles in disulfide bond formation and sperm maturation

Thioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization of protein and interprotein disulfide bonds and localized this function to its thiol domain. TGR targets include proteins that form structural components of the sperm, including glutathione peroxidase GPx4/PHGPx. Together, TGR and GPx4 can serve as a novel disulfide bond formation system. Both enzymes contain a catalytic selenocysteine consistent with the role of selenium in male reproduction.     

A novel monothiol glutaredoxin (Grx4) from Escherichia coli can serve as a substrate for thioredoxin reductase

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750-2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.     

Structural Analysis of Glutaredoxin Domain of Mus musculus Thioredoxin Glutathione Reductase

Thioredoxin glutathione reductase (TGR) is a member of the mammalian thioredoxin reductase family that has a monothiol glutaredoxin (Grx) domain attached to the thioredoxin reductase module. Here, we report a structure of the Grx domain of mouse TGR, determined through high resolution NMR spectroscopy to the final backbone RMSD value of 0.48±0.10 Å. The structure represents a sandwich-like molecule composed of a four stranded β-sheet flanked by five α–helixes, with the CxxS active motif located on the catalytic loop. We structurally characterized the glutathione-binding site in the protein and describe sequence and structural relationships of the domain with glutaredoxins. The structure illuminates a key functional center that evolved in mammalian TGRs to act in thiol-disulfide reactions. Our study allows us to hypothesize that Cys105 might be functionally relevant for TGR catalysis. In addition, the data suggest that the N-terminus of Grx acts as a possible regulatory signal also protecting the protein active site from unwanted interactions in cellular cytosol.     

The thioredoxin reductase-glutaredoxins-ferredoxin crossroad pathway for selenate tolerance in Synechocystis PCC6803

Most organisms use two systems to maintain the redox homeostasis of cellular thiols. In the thioredoxin (Trx) system, NADPH sequentially reduces thioredoxin reductases (NTR), Trxs and protein disulfides. In the glutaredoxin (Grx) system, NADPH reduces the glutathione reductase enzyme occurring in most organisms, glutathione, Grxs, and protein disulfides or glutathione-protein mixed disulfides. As little is known concerning these enzymes in cyanobacteria, we have undertaken their analysis in the model strain Synechocystis PCC6803. We found that Grx1 and Grx2 are active, and that Grx2 but not Grx1 is crucial to tolerance to hydrogen peroxide and selenate. We also found that Synechocystis has no genuine glutathione reductase and uses NTR as a Grx electron donor, in a novel integrative pathway NADPH-NTR-Grx1-Grx2-Fed7 (ferredoxin 7), which operates in protection against selenate, the predominant form of selenium in the environment. This is the first report on the occurrence of a physical interaction between a Grx and a Fed, and of an electron transfer between two Grxs. These findings are discussed in terms of the (i) selectivity of Grxs and Feds ( Synechocystis possesses nine Feds), (ii) crucial importance of NTR for cell fitness and (iii) resistance to selenate, in absence of a Thauera selenatis -like selenate reductase.     

¹H, ¹³C, and ¹⁵N NMR resonance assignments of reduced full length and shortened forms of the Grx domain of Mus musculus TGR

Two forms of the glutaredoxin (Grx) domain (full length Grx domain and short Grx lacking the N-terminal region) of Mus musculus thioredoxin glutathione reductase (TGR) were isotopically labelled with ¹⁵N and ¹³C isotopes, expressed and purified to homogeneity. We report here the ¹H, ¹³C and ¹⁵N NMR assignment for both Grx forms of this mouse TGR. This investigation represents the first NMR analysis of a mammalian TGR.     

Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins

Inorganic sulfate (SO42-, S+VI) is reduced in vivo to sulfite (SO32-, S+IV) via phosphoadenylylsulfate (PAPS) reductase. Escherichia coli lacking glutathione reductase and glutaredoxins (gor-grxA-grxB-grxC-) barely grows on sulfate. We found that incubation of PAPS reductase with oxidized glutathione leads to enzyme inactivation with simultaneous formation of a mixed disulfide between glutathione and the active site Cys-239. A newly developed method based on thiol-specific fluorescent alkylation and gel electrophoresis showed that glutathionylated PAPS reductase is reduced by glutaredoxins via a monothiol mechanism. This glutathionylated species was also observed in poorly growing gor-grxA-grxB-grxC- cells expressing inactive glutaredoxin 2 (Grx2) C9S/C12S. However, it was absent in better growing cells expressing monothiol Grx2 C12S or wild type Grx2. Reversible glutathionylation may thus regulate the activity of PAPS reductase in vivo.     

Thioredoxin glutathione reductase as a novel drug target: evidence from Schistosoma japonicum

Background

Schistosomiasis remains a major public health concern affecting billions of people around the world. Currently, praziquantel is the only drug of choice for treatment of human schistosomiasis. The emergence of drug resistance to praziquantel in schistosomes makes the development of novel drugs an urgent task. Thioredoxin glutathione reductase (TGR) enzymes in Schistosoma mansoni and some other platyhelminths have been identified as alternative targets. The present study was designed to confirm the existense and the potential value of TGR as a target for development of novel antischistosomal agents in Schistosoma japonicum, a platyhelminth endemic in Asia.

Methods and Findings

After cloning the S. japonicum TGR (SjTGR) gene, the recombinant SjTGR selenoprotein was purified and characterized in enzymatic assays as a multifunctional enzyme with thioredoxin reductase (TrxR), glutathione reductase (GR) and glutaredoxin (Grx) activities. Immunological and bioinformatic analyses confirmed that instead of having separate TrxR and GR proteins in mammalian, S. japonicum only encodes TGR, which performs the functions of both enzymes and plays a critical role in maintaining the redox balance in this parasite. These results were in good agreement with previous findings in Schistosoma mansoni and some other platyhelminths. Auranofin, a known inhibitor against TGR, caused fatal toxicity in S. japonicum adult worms in vitro and reduced worm and egg burdens in S. japonicum infected mice.

Conclusions

Collectively, our study confirms that a multifunctional enzyme SjTGR selenoprotein, instead of separate TrxR and GR enzymes, exists in S. japonicum. Furthermore, TGR may be a potential target for development of novel agents against schistosomes. This assumption is strengthened by our demonstration that the SjTGR is an essential enzyme for maintaining the thiol-disulfide redox homeostasis of S. japonicum.     

Solution structure of Escherichia coli glutaredoxin-2 shows similarity to mammalian glutathione-S-transferases.

Glutaredoxin 2 (Grx2) from Escherichia coli is distinguished from other glutaredoxins by its larger size, low overall sequence identity and lack of electron donor activity with ribonucleotide reductase. However, catalysis of glutathione (GSH)-dependent general disulfide reduction by Grx2 is extremely efficient. The high-resolution solution structure of E. coli Grx2 shows a two-domain protein, with residues 1 to 72 forming a classical "thioredoxin-fold" glutaredoxin domain, connected by an 11 residue linker to the highly helical C-terminal domain, residues 84 to 215. The active site, Cys9-Pro10-Tyr11-Cys12, is buried in the interface between the two domains, but Cys9 is solvent-accessible, consistent with its role in catalysis. The structures reveal the hither to unknown fact that Grx2 is structurally similar to glutathione-S-transferases (GST), although there is no obvious sequence homology. The similarity of these structures gives important insights into the functional significance of a new class of mammalian GST-like proteins, the single-cysteine omega class, which have glutaredoxin oxidoreductase activity rather than GSH-S-transferase conjugating activity. E. coli Grx 2 is structurally and functionally a member of this new expanding family of large glutaredoxins. The primary function of Grx2 as a GST-like glutaredoxin is to catalyze reversible glutathionylation of proteins with GSH in cellular redox regulation including stress responses.     

Identification of thioredoxin glutathione reductase inhibitors that kill cestode and trematode parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 μM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites.     

Analysis of the inhibition of mammalian thioredoxin, thioredoxin reductase, and glutaredoxin by cis-diamminedichloroplatinum (II) and its major metabolite, the glutathione-platinum complex.

Several studies have demonstrated a correlation between cellular toxicity of cis-diamminedichloroplatinum (II) (cisplatin, CDDP) and inhibited intracellular activity of the thioredoxin system, i.e., thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH. Conversely, increased cellular activity of the Trx system confers resistance to CDDP. In this study, we have analyzed the interaction of CDDP with Trx and TrxR in order to clarify the mechanism. The inhibition with time-dependent kinetics by CDDP of NADPH-reduced (but not oxidized) TrxR was irreversible, strongly suggesting covalent modification of the reduced selenocysteine-containing active site. Assuming second order kinetics, the rate constant of TrxR inhibition by CDDP was 21 +/- 3 M(-1) x s(-1). Transplatin was found to be an even more efficient inhibitor, with a second order rate constant of 84 +/- 22 M(-1) x s(-1), whereas carboplatin (up to 1 mM) gave no inhibition of the enzyme under the same conditions. Escherichia coli Trx or human or bacterial glutaredoxin (Grx) activities were in comparison only slightly or not at all inhibited by either CDDP, transplatin, or carboplatin. However, glutaredoxins were found to be inhibited by the purified glutathione adduct of cisplatin, bis-(glutathionato)platinum(II) (GS-Platinum complex, GS-Pt), with an IC50 = 350 microM in the standard beta-hydroxyethyl disulfide-coupled assay for human Grx. Also the mammalian Trx system was inhibited by GS-Pt with similar efficiency (IC(50) = 325 microM), whereas neither the E. coli Trx system nor glutathione reductase were inhibited. Formation of GS-Pt is a major route for cellular elimination of CDDP. The fact that GS-Pt inhibits the mammalian Trx as well as Grx systems shows that CDDP may exert effects at several stages of its metabolism, including after conjugation with GSH, which are intimately linked with the cellular disulfide/dithiol redox regulatory systems.     

Unique gene organization of thioredoxin and thioredoxin reductase in Mycobacterium leprae

The thioredoxin system comprising thioredoxin (Trx), thioredoxin reductase (TR) and NADPH operates via redox-active disulphides and provides electrons for a wide variety of different metabolic processes in prokaryotic and eukaryotic cells. Thioredoxin is also a general protein disulphide reductase involved in redox regulation. In bacteria, the Trx and TR proteins previously identified were encoded by separate genes (trxA and trxB). In this study, we report a novel genomic organization of TR and Trx in mycobacteria and show that at least three modes of organization of TR and Trx genes can exist within a single bacterial genus: (i) in the majority of mycobacterial strains the genes coding for TR and Trx are located on separate sites of the genome; (ii) interestingly, in all pathogenic Mycobacterium tuberculosis complex mycobacteria both genes are found on the same locus, overlapping in one nucleotide; (iii) in the pathogen Mycobacterium leprae, TR and Trx are encoded by a single gene. Sequence analysis of the M. leprae gene demonstrated that the N-terminal part of the protein corresponds to TR and the C-terminal part to Trx. A corresponding single protein product of approximately 49 kDa was detected in cell extracts of M. leprae. These findings demonstrate the very unusual phenomenon of a single gene coding for both the substrate (thioredoxin) and the enzyme (thioredoxin reductase), which seems to be unique to M. leprae.     

The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 A resolution.

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs. We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis. The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R-factor of 18.9% for 53,868 reflections with F > or = sigma F between 8.0 and 2.3 A resolution. The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules. The accuracy and geometry of the enzyme model is improved with respect to the C. fasciculata enzyme model. The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli. Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.