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Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase

2001; Elsevier BV; Volume: 276; Issue: 14 Linguagem: Inglês

10.1074/jbc.m010352200

1083-351X

Matthias Dormeyer, Nina Reckenfelderbäumer, Heike Lüdemann, R. Luise Krauth‐Siegel,

Redox biology and oxidative stress

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. bruceiribonucleotide reductase with a Km 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 Km value of T. brucei ribonucleotide reductase for T. bruceitryparedoxin is about 4 μm. 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. 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. bruceiribonucleotide reductase with a Km 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 Km value of T. brucei ribonucleotide reductase for T. bruceitryparedoxin is about 4 μm. 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. large and small subunit, respectively, of ribonucleotide reductase dithioerythritol 5,5′-dithiobis(2-nitrobenzoate) (mono)glutathionylspermidine trypanothione [N1,N8-bis(glutathionyl)spermidine] trypanothione disulfide (oxidized trypanothione) trypanothione reductase high pressure liquid chromatography Ribonucleotide reductases (E.C. 1.17.4.1) catalyze the rate-limiting step in the de novo synthesis of DNA precursors and thus are key enzymes for the replication of an organism (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (617) Google Scholar). African trypanosomes possess a typical eukaryotic class I ribonucleotide reductase (2Hofer A. Schmidt P.P. Gräslund A. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6959-6964Crossref PubMed Scopus (50) Google Scholar, 3Dormeyer M. Schöneck R. Dittmar G.A.G. Krauth-Siegel R.L. FEBS Lett. 1997; 414: 449-453Crossref PubMed Scopus (19) Google Scholar, 4Hofer A. Ekanem J.T. Thelander L. J. Biol. Chem. 1998; 273: 34098-34104Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The genes encoding theTrypanosoma brucei R1 and R21 proteins have been cloned and overexpressed in Escherichia coli. Class I enzymes are tetrameric proteins composed of two R1 and R2 molecules each. The large R1 protein harbors the active site, as well as regulatory sites, and the small R2 protein contains a μ-oxo-bridged diiron cluster, which represents the Fe(III)–O2−–Fe(III) cofactor in all eukaryotic ribonucleotide reductases, and a tyrosyl radical essential for catalysis (1Jordan A. Reichard P. Annu. Rev. Biochem. 1998; 67: 71-98Crossref PubMed Scopus (617) Google Scholar, 5Sjöberg B.-M. Eckstein F. Lilley D.M.J. Nucleic Acids and Molecular Biology. 9. Springer-Verlag, Berlin1995: 192-221Google Scholar). T. bruceiribonucleotide reductase is regulated via the R2 subunit. Whereas the R1 protein is present throughout the life cycle of the parasite, the R2 protein is not found in cell cycle-arrested short stumpy trypanosomes (6Breidbach T. Krauth-Siegel R.L. Steverding D. FEBS Lett. 2000; 473: 212-216Crossref PubMed Scopus (12) Google Scholar). Reduction of the 2′-OH group of ribonucleoside diphosphates to the corresponding deoxynucleotides requires external electron donors. For class I enzymes, small thiol proteins with an active site CXXC motif like thioredoxin (CGPC) and glutaredoxin (CPYC) are well known hydrogen donors (7Holmgren A. J. Biol. Chem. 1989; 264: 13963-13966Abstract Full Text PDF PubMed Google Scholar, 8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). Oxidized thioredoxin is subsequently reduced by thioredoxin reductase at the expense of NADPH (8Holmgren A. Annu. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar). The dithiol form of glutaredoxin is spontaneously regenerated by glutathione. Glutathione disulfide formed in the reaction is then reduced by NADPH and glutathione reductase (9Holmgren A. J. Biol. Chem. 1979; 254: 3664-3671Abstract Full Text PDF PubMed Google Scholar, 10Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar). Trypanosomatids are the causative agents of tropical diseases such as South American Chagas' disease (Trypanosoma cruzi), African sleeping sickness (T. brucei rhodesiense and T. brucei gambiense), Nagana cattle disease (T. congolense andT. brucei brucei), and the three manifestations of Leishmaniasis. All these parasitic protozoa have in common that the ubiquitous glutathione/glutathione reductase system is replaced by a trypanothione/trypanothione reductase system. Monoglutathionylspermidine (Gsp) and trypanothione (N1,N8-bis(glutathionyl)spermidine; T(SH)2) are the main low molecular mass thiols and are responsible for the redox balance of the cell (11Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (687) Google Scholar, 12Krauth-Siegel R.L. Schöneck R. FASEB. J. 1995; 9: 1138-1146Crossref PubMed Scopus (73) Google Scholar). These glutathionylspermidine conjugates are kept reduced by the flavoenzyme trypanothione reductase (TS2 + NADPH + H+ → T(SH)2 + NADP), an essential enzyme of the parasite (13Krieger S. Schwarz W. Ariyanayagam M.R. Fairlamb A.H. Krauth-Siegel R.L. Clayton C. Mol. Microbiol. 2000; 35: 542-552Crossref PubMed Scopus (312) Google Scholar,14Krauth-Siegel R.L. Coombs G. Parasitol. Today. 1999; 15: 404-409Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Trypanothione spontaneously reduces dehydroascorbate (15Krauth-Siegel R.L. Lüdemann H. Mol. Biochem. Parasitol. 1996; 80: 203-208Crossref PubMed Scopus (53) Google Scholar) and hydrogen peroxide (16Carnieri E.G.S. Moreno S.N.J. Docampo R. Mol. Biochem. Parasitol. 1993; 1993: 79-86Crossref Scopus (70) Google Scholar). The latter reaction is catalyzed by an enzyme cascade composed of trypanothione, trypanothione reductase, tryparedoxin, and a tryparedoxin peroxidase (17Nogoceke E. Gommel D.U. Kies M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (263) Google Scholar, 18Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (183) Google Scholar). Tryparedoxin is a 16-kDa protein with an active site WCPPC motif (19Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar, 20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). The gene encoding the T. brucei protein has been cloned and overexpressed. Tryparedoxin functions as a trypanothione-dependent thiol-disulfide oxidoreductase with catalytic properties intermediate between those of classical thioredoxins and glutaredoxin (20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). The known dependence of eukaryotic ribonucleotide reductases on external thiols prompted us to study whether trypanothione is able to provide the electrons for the parasite synthesis of deoxyribonucleotides. Here we will show that the trypanosomatid-specific dithiol trypanothione, in contrast to the monothiol glutathione, is a direct donor of reducing equivalents forT. brucei ribonucleotide reductase and that the reaction is catalyzed by tryparedoxin. [3H]GDP was purchased from Amersham Pharmacia Biotech, GDP and dTTP were from Sigma, trypanothione disulfide (TS2) and glutathionylspermidine disulfide (Gspox) were from Bachem, and NaBH4 was from Fluka. All chemicals were of the highest available purity. C18 cartridges were obtained from Millipore, and the Aminex A9 anion exchange resin was from Bio-Rad. The plasmids encodingT. brucei R1 and R2 were kindly provided by Drs. Anders Hofer and Lars Thelander, Umeå, Sweden. RecombinantT. brucei tryparedoxin (20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar), ribonucleotide reductase (2Hofer A. Schmidt P.P. Gräslund A. Thelander L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6959-6964Crossref PubMed Scopus (50) Google Scholar,3Dormeyer M. Schöneck R. Dittmar G.A.G. Krauth-Siegel R.L. FEBS Lett. 1997; 414: 449-453Crossref PubMed Scopus (19) Google Scholar), and T. cruzi trypanothione reductase (21Sullivan F.X. Walsh C.T. Mol. Biochem. Parasitol. 1991; 44: 145-147Crossref PubMed Scopus (102) Google Scholar, 22Krauth-Siegel R.L. Sticherling C. Jöst I. Walsh C.T. Pai E.F. Kabsch W. Lantwin C.B. FEBS Lett. 1993; 317: 105-108Crossref PubMed Scopus (30) Google Scholar) were purified as described. Alkaline phosphatase from calf intestine was purchased from Roche Molecular Biochemicals. Human glutathione reductase was a kind gift of Dr. R. Heiner Schirmer, Biochemie-Zentrum Heidelberg. Ribonucleotide reductase activity was determined from the rate of conversion of [3H]GDP into [3H]dGDP essentially as described for CDP reduction (23Engström Y. Eriksson S. Thelander L. Åkerman M. Biochemistry. 1979; 18: 2941-2948Crossref PubMed Scopus (119) Google Scholar). The assay mixture contained, in a total volume of 200 μl, 50 mm Hepes, pH 7.6, 500 μm GDP (including 1.25 μCi [3H]GDP), 100 μm dTTP, 100 mm KCl, 6.4 mm MgCl2, and variable concentrations of thiols and tryparedoxin. In the standard assay 1 unit of T. bruceiR1 (about 40 μg of protein ≈ 0.4 nmol) with a 5-fold molar excess of R2 was used (1 unit corresponds to 1 nmol of dGDP formation/min). The reaction mixture was incubated at 37 °C for 10 and 20 min, respectively, the reaction was stopped by boiling for 10 min, and the precipitated protein was removed by centrifugation. The reaction components were dephosphorylated by 45-min incubation with 10 units of alkaline phosphatase. Guanosine, deoxyguanosine, and guanine were separated isocratically by HPLC on an Aminex A9 column (250 × 4 mm) in 100 mm ammonium borate, pH 8.3, and quantified by scintillation counting (24Willing A. Follmann H. Auling G. Eur. J. Biochem. 1988; 170: 603-611Crossref PubMed Scopus (173) Google Scholar). The thiols were generated in situ in the ribonucleotide reductase reaction mixture containing all components except R1, R2, and radiolabeled GDP. Glutathione disulfide was reduced by 200 milliunits of human glutathione reductase, glutathionylspermidine disulfide, and trypanothione disulfide by 200 milliunits of T. cruzi TR in the presence of a 2.5-fold molar excess of NADPH. The mixture was incubated for 15 min at 37 °C, and the ribonucleotide reductase reaction was started by adding R1, R2, and [3H]GDP. 10 mm trypanothione disulfide in 1 ml of water was incubated on ice with 100 mm NaBH4 for 1 h. The solution was acidified with 1 m HCl to pH 3.0 to prevent reoxidation of the thiol after decomposition of excess hydride. A C18 cartridge was washed with 4 ml of acetonitrile followed by 10 ml of water. The reaction mixture was applied, and the cartridge was washed with 3 ml of 0.1% trifluoroacetic acid. Trypanothione was eluted with 1.5 ml of 80% acetonitrile in 0.1% trifluoroacetic acid, lyophilized, dissolved in 50 mm Hepes, pH 7.6, to a final concentration of 25 mm and, used immediately. The concentration of free thiols was determined by reaction with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; see Ref. 25Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21218) Google Scholar). In a final volume of 100 μl 50 μm tryparedoxin were incubated with 1 mm T(SH)2 in 50 mm Hepes, pH 7.6, for 15 min under argon atmosphere. 3 μl of 200 mmiodoacetamide in water were added. After a 60-min incubation at room temperature in the dark, the reaction was stopped by adding 15 μl of 200 mm DTE in water. A control reaction contained tryparedoxin and iodoacetamide but no T(SH)2 and was not stopped by DTE. The low molecular mass components were removed by centrifugation in a Centricon 3 concentrator (Millipore), and the modified protein was washed several times with 50 mm Hepes, pH 7.6. This procedure resulted in a homogeneous protein sample (see below) that represents tryparedoxin specifically modified at the first cysteine residue (Cys-40) of the WCPPC motif as described forCrithidia fasciculata tryparedoxin (19Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar). For the alkylation of both active site cysteinyl residues, the reaction was carried out in the presence of 6 m guanidinium chloride. Oxidized, reduced, mono-, and bis-carboxamidomethylated tryparedoxin were separated by HPLC on a VYDAAC 208 TP column at a flow rate of 0.2 ml/min by a linear gradient from 38.5 to 45.5% acetonitrile in 0.1% trifluoroacetic acid within 1 h. The proteins were detected at 214 nm. The content of free thiol groups was determined with DTNB (25Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21218) Google Scholar). The thiol content of unmodified tryparedoxin was determined after reduction with NaBH4 at room temperature for 5 min. HCl was added to destroy excess NaBH4, and an aliquot of the sample was immediately analyzed for free thiol groups. Formation of [3H]dGDP from [3H]GDP by T. brucei ribonucleotide reductase was followed in the presence of trypanothione, glutathionylspermidine, glutathione, and the nonphysiological dithiol DTE. T(SH)2, Gsp, and GSH were generated and kept reduced by NADPH/TR and NADPH/glutathione reductase, respectively. Control assays revealed that the activity of ribonucleotide reductase is only slightly affected by millimolar concentrations of NADPH and NADP (data not shown). Trypanothione is an efficient reductant of the parasite enzyme. At a fixed concentration of 2 mm thiol groups, the activity of ribonucleotide reductase with trypanothione amounts to 30% of that observed with DTE (Fig. 1, light gray columns). In contrast, the monothiol Gsp showed very low activity, and GSH was completely inactive at this concentration. TheKm values of T. brucei ribonucleotide reductase for T(SH)2 and DTE were determined by varying the concentration of the dithiol from 0.5 to 4 mm and 2 to 12 mm, respectively. The reactions followed Michaelis-Menten kinetics and yielded Km values of 2.1 ± 0.4 mm for T(SH)2 and 6.9 ± 1.2 mm for DTE (Table I). Because the external electron donors for ribonucleotide reductase interact with the R1 protein (26Mao S.S. Holler T.P., Yu, G.X. Bollinger Jr., J.M. Booker S. Johnston M.I. Stubbe J. Biochemistry. 1992; 31: 9733-9743Crossref PubMed Scopus (201) Google Scholar, 27Eriksson M. Uhlin U. Ramaswamy S. Ekberg M. Regnström K. Sjöberg B.-M. Eklund H. Structure. 1997; 5: 1077-1092Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) the assays contained a molar excess of R2, and the specific activity refers to the amount of R1 protein. The maximum reaction rates were calculated by extrapolating to saturating concentrations of dithiol. The Vmax value of ribonucleotide reductase with DTE was about 6-fold higher than that with T(SH)2 (Table I).Table IKinetic parameters of T. brucei ribonucleotide reductaseKmVmaxμmnmol/min · mgDTE6900 ± 120060T(SH)22100 ± 4009.6Tryparedoxin1-aThe assays contained a constant concentration of 2.5 mm trypanothione.3.7 ± 0.524The assays were carried out as described under "Experimental Procedures," and the kinetic data were derived from Lineweaver-Burk plots. Mean values of at least three independent measurements, together with the standard deviations, are given.1-a The assays contained a constant concentration of 2.5 mm trypanothione. Open table in a new tab The assays were carried out as described under "Experimental Procedures," and the kinetic data were derived from Lineweaver-Burk plots. Mean values of at least three independent measurements, together with the standard deviations, are given. The activity of T. brucei ribonucleotide reductase with DTE, T(SH)2, Gsp, and GSH was followed in the absence and presence of T. brucei tryparedoxin. The protein stimulated the rate of dGDP formation in the presence of all four thiols. At 1 mm low molecular mass dithiol, the trypanothione/tryparedoxin couple yielded about 50% activity compared with DTE/tryparedoxin. With tryparedoxin in the reaction mixture, the monothiols glutathionylspermidine and glutathione also caused a pronounced dGDP formation (Fig. 1, dark gray columns). When the ribonucleotide reductase activities in the presence of thiol and tryparedoxin were corrected for the respective activity with the thiol alone, DTE (0.185 nmol/min), trypanothione (0.21 nmol/min), and glutathionylspermidine (0.205 nmol/min) resulted in nearly identical rates of GDP reduction. Only with GSH (0.1 nmol/min) the activity of ribonucleotide reductase was significantly lower in accordance with a weak reduction of tryparedoxin by GSH (20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). As shown in Fig. 1, at millimolar concentrations, trypanothione is an efficient direct hydrogen donor for ribonucleotide reductase. At lower T(SH)2 concentrations, stimulation of the reaction by tryparedoxin becomes pronounced. For instance, at 100 μmtrypanothione, 4 μm tryparedoxin increased dGDP formation by a factor of 14 (data not shown). The Km value of T. brucei ribonucleotide reductase for tryparedoxin was determined in the presence of a constant concentration of 2.5 mm T(SH)2. The dependence of the reaction rate on the tryparedoxin concentration showed saturation kinetics and yielded an apparent Km value of 3.7 ± 0.5 μm (Fig.2). Tryparedoxin was carboxamidomethylated with iodoacetamide under nondenaturing conditions that, in analogy toE. coli thioredoxin (28Kallis G.-B. Holmgren A. J. Biol. Chem. 1980; 255: 10261-10265Abstract Full Text PDF PubMed Google Scholar) and C. fasciculatatryparedoxin (19Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar), should result in the exclusive modification of Cys-40, the first cysteine residue of the WCPPC motif. Analysis of the modified protein with Ellman's reagent yielded a total of 5 nmol of thiol groups per 6 nmol of protein whereas the control contained 8.6 nmol of free thiol groups per 5.6 nmol of protein. The relatively low thiol content of the control sample may be because of formation of covalent dimers that were observed when storing the NaBH4-reduced protein at pH 7.6 (not shown). HPLC analysis of the carboxamidomethylated protein revealed a single peak in accordance with the specific modification of one cysteine residue. As expected, the monoalkylated tryparedoxin did not catalyze the trypanothione-dependent reduction of GDP by ribonucleotide reductase (Table II).Table IIEffect of carboxamidomethylation of T. brucei tryparedoxin on ribonucleotide reductase activityRibonucleotide reductaserowsep="1" nmol dGDP/minT(SH)20.04T(SH)2 + tryparedoxin0.33T(SH)2 + monoalkylated tryparedoxin0.06All reaction mixtures contained 250 μm trypanothione. The tryparedoxin concentration was 4 μm. The assays were performed as described under "Experimental Procedures." Open table in a new tab All reaction mixtures contained 250 μm trypanothione. The tryparedoxin concentration was 4 μm. The assays were performed as described under "Experimental Procedures." The effect of TS2 on the activity ofT. brucei ribonucleotide reductase was studied in the reaction with trypanothione as sole reductant and in the trypanothione/tryparedoxin system (Fig.3). Trypanothione disulfide showed only a minor effect on the activity of ribonucleotide reductase. At 1 mm trypanothione, 2.5 mm trypanothione disulfide diminished the rate of GDP reduction by about 40% (Fig.3 a). In contrast, the tryparedoxin-mediated reaction proved to be much more sensitive. 2.5 mm TS2 in the presence of 1 mm T(SH)2 and 10 μmtryparedoxin inhibited the rate of deoxyribonucleotide formation by 90% (Fig. 3 b). The residual activity of ribonucleotide reductase was identical with that observed with trypanothione alone indicating that it is tryparedoxin and not ribonucleotide reductase that is strongly regulated by the thiol/disulfide ratio of trypanothione. The IC50 value of tryparedoxin for trypanothione disulfide is about 50 μm in the presence of 1 mm T(SH)2. The pronounced sensitivity of tryparedoxin toward trypanothione disulfide also became evident when NADPH and trypanothione reductase were added to the assays (Fig. 3, a and b,second column). In the trypanothione/ribonucleotide reductase assay the rate of dGDP formation increased by only 10% whereas in the trypanothione/tryparedoxin/ribonucleotide reductase system the activity was doubled. The sample of trypanothione used in these experiments contained about 4% disulfide as revealed by an end point determination in a trypanothione reductase assay. This corresponds to a concentration of 40 μm TS2at the beginning, in addition to trypanothione disulfide formed during the reaction, and explains the pronounced effect of trypanothione reductase/NADPH. The discovery of the trypanothione system in Kinetoplastida raised the question as to the specific functions of the dithiol. The pivotal role of trypanothione in the antioxidant defense mechanisms of the parasites is well established (11Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (687) Google Scholar, 13Krieger S. Schwarz W. Ariyanayagam M.R. Fairlamb A.H. Krauth-Siegel R.L. Clayton C. Mol. Microbiol. 2000; 35: 542-552Crossref PubMed Scopus (312) Google Scholar, 14Krauth-Siegel R.L. Coombs G. Parasitol. Today. 1999; 15: 404-409Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 15Krauth-Siegel R.L. Lüdemann H. Mol. Biochem. Parasitol. 1996; 80: 203-208Crossref PubMed Scopus (53) Google Scholar, 16Carnieri E.G.S. Moreno S.N.J. Docampo R. Mol. Biochem. Parasitol. 1993; 1993: 79-86Crossref Scopus (70) Google Scholar, 17Nogoceke E. Gommel D.U. Kies M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (263) Google Scholar). As shown here, trypanothione is also involved in the parasite synthesis of DNA precursors (Fig. 4). The dithiol serves as direct donor of reducing equivalents for T. bruceiribonucleotide reductase. In contrast, monoglutathionylspermidine and glutathione result in very low and no activity, respectively, in accordance with other ribonucleotide reductases where DTE and lipoate are hydrogen donors whereas monothiols are inactive (29Thelander L. Reichard P. Annu. Rev. Biochem. 1979; 48: 133-158Crossref PubMed Scopus (909) Google Scholar). The ability of trypanothione, but not of glutathione, to reduce ribonucleotide reductase directly is not related to the redox potentials of the thiols that are very similar (−242 and −230 mV for T(SH)2 and GSH, respectively; see Ref. 11Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (687) Google Scholar). In contrast, the pK values of the thiols differ significantly. A pK value of 7.4 has been reported for trypanothione, which is more than one pH unit lower than the pK value of 8.7 of GSH (30Moutiez M. Meziane-Sherif D. Aumercier M. Sergheraert C. Tartar A. Chem. Pharm. Bull. ( Tokyo ). 1994; 42: 2641-2644Crossref Scopus (45) Google Scholar). Because second order rate constants for thiol-disulfide exchanges exhibit an optimum when the thiol pK value is equal to the pH value of the solution, T(SH)2 is expected to be much more reactive than GSH under physiological conditions. In addition, as reductants for intramolecular disulfides as in the R1 protein, dithiols are kinetically superior to monothiols (31Gilbert H.F. Adv. Enzymol. Relat. Areas Mol. Biol. 1990; 63: 69-172PubMed Google Scholar). Trypanothione is the first example of a natural low molecular mass dithiol that is a direct reductant of ribonucleotide reductase. The trypanothione-dependent synthesis of deoxyribonucleotides by T. brucei ribonucleotide reductase is catalyzed by T. brucei tryparedoxin. This thioredoxin-like protein has been found exclusively in trypanosomatids, and its first elucidated role was as component of a trypanothione-dependent peroxidase cascade (17Nogoceke E. Gommel D.U. Kies M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (263) Google Scholar, 18Flohé L. Hecht H.J. Steinert P. Free Radic. Biol. Med. 1999; 27: 966-984Crossref PubMed Scopus (183) Google Scholar, 19Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar). The apparent Km value of T. bruceiribonucleotide reductase for T. brucei tryparedoxin (3.7 μm) is higher than those of E. coliribonucleotide reductase for glutaredoxins 1 and 3 (0.13 and 0.35 μm, respectively) (32Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar). It is comparable with that for thioredoxin (1.3 μm) in the E. coli system (10Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar). The maximum activity of recombinant T. bruceiribonucleotide reductase in the trypanothione/tryparedoxin system is 24 nmol/min·mg R1. This value is in the same order of magnitude as the varying activities reported for the E. coli enzyme with thioredoxins 1 or 2 and glutaredoxin 1 (10Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar, 33Miranda-Vizuete A. Damdimopoulos E. Gustafsson J.-Å. Spyrou G. J. Biol. Chem. 1997; 272: 30841-30847Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and thus is significantly higher than that of E. coli ribonucleotide reductase with glutaredoxin 3 as hydrogen donor where theVmax is only 5% that of glutaredoxin 1 (32Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar). It is not possible to determine the Km value of tryparedoxin for T(SH)2 using formation of dGDP as the indicator reaction. The reaction catalyzed by ribonucleotide reductase is three orders of magnitude slower than the preceding reduction of the enzyme by tryparedoxin assuming that the latter reaction occurs at a rate similar to the reduction of tryparedoxin peroxidase by tryparedoxin (17Nogoceke E. Gommel D.U. Kies M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (263) Google Scholar, 34Montemartini M. Kalisz H.M. Hecht H.-J. Steinert P. Flohé L. Eur. J. Biochem. 1999; 264: 516-524Crossref PubMed Scopus (67) Google Scholar). Km values of different tryparedoxins for T(SH)2 between 30 and 150 μm have been estimated using the tryparedoxin peroxidase/hydroperoxide system or glutathione disulfide (oxidized glutathione) as the final electron acceptor (19Gommel D.U. Nogoceke E. Morr M. Kiess M. Kalisz H.M. Flohé L. Eur. J. Biochem. 1997; 248: 913-918Crossref PubMed Scopus (100) Google Scholar, 20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar, 35Montemartini M. Kalisz H.M. Kiess M. Nogoceke E. Singh M. Steinert P. Flohé L. Biol. Chem. 1998; 379: 1137-1142Crossref PubMed Scopus (35) Google Scholar). The trypanothione concentration in T. brucei is 400–800 μm (11Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (687) Google Scholar), which should be adequate to keep tryparedoxin predominantly in the reduced state. In addition, tryparedoxin is a very abundant protein. In the insect parasite C. fasciculata, it represents 5% of the total soluble protein of the cell (17Nogoceke E. Gommel D.U. Kies M. Kalisz H.M. Flohé L. Biol. Chem. 1997; 378: 827-836Crossref PubMed Scopus (263) Google Scholar). Taken together, these data indicate that reduction of ribonucleotide reductase by the trypanothione/tryparedoxin system is not a limiting factor in the parasite synthesis of deoxyribonucleotides. The tryparedoxin-mediated activity of ribonucleotide reductase was highest with DTE and trypanothione, but in the presence of tryparedoxin the monothiols Gsp and GSH, which fail as direct hydrogen donors, also yielded a significant dGDP formation. The increase of ribonucleotide reductase activity caused by tryparedoxin was comparable for trypanothione and monoglutathionylspermidine but much lower with GSH as the hydrogen donor. Because the glutathionylspermidine conjugates are the main low molecular mass thiols in the parasites (11Fairlamb A.H. Cerami A. Annu. Rev. Microbiol. 1992; 46: 695-729Crossref PubMed Scopus (687) Google Scholar) they are most probably the physiological electron donors in the parasite synthesis of DNA precursors. Trypanothione disulfide proved to be a powerful inhibitor of the tryparedoxin-mediated ribonucleotide reduction. At a T(SH)2/TS2 ratio of 10:1, the activity ofT. brucei ribonucleotide reductase is lowered by more than 60%. Inhibition of tryparedoxin by TS2 may be a physiological control mechanism. For E. coli glutaredoxin a respective observation has been made. Glutaredoxin is strongly inhibited in the presence of glutathione disulfide (oxidized glutathione) (10Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar) indicating a relationship between the rate of DNA synthesis and the redox state of the cell. In the reaction with ribonucleotide reductase, tryparedoxin resembles mechanistically glutaredoxin. The ultimate reductant is a low molecular mass thiol, and the reaction is inhibited by the disulfide form of the respective thiol (10Holmgren A. J. Biol. Chem. 1979; 254: 3672-3678Abstract Full Text PDF PubMed Google Scholar). In contrast, with respect to the protein sequence, as well as thiol-disulfide exchange reactions, tryparedoxin is more similar to thioredoxins (20Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar). Obviously, the parasite dithiol protein has properties intermediate between those of classical thioredoxins and glutaredoxins. In all organisms with class I ribonucleotide reductases investigated so far different hydrogen donor systems occur simultaneously. For instance, E. coli contains two thioredoxins and two glutaredoxins that are able to deliver electrons for ribonucleotide reductase (32Åslund F. Ehn B. Miranda-Vizuete A. Pueyo C. Holmgren A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9813-9817Crossref PubMed Scopus (164) Google Scholar, 33Miranda-Vizuete A. Damdimopoulos E. Gustafsson J.-Å. Spyrou G. J. Biol. Chem. 1997; 272: 30841-30847Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Recently we have cloned the gene encoding a classical thioredoxin from T. brucei (36Reckenfelderbäumer N. Lüdemann H. Schmidt H. Steverding D. Krauth-Siegel R.L. J. Biol. Chem. 2000; 275: 7547-7552Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The recombinant protein is also a hydrogen donor for the trypanosomal ribonucleotide reductase when applied together with DTE or NADPH and human thioredoxin reductase. 2H. Schmidt and R. L. Krauth-Siegel, unpublished results. The thioredoxin gene is expressed throughout the life cycle of T. brucei, but the protein concentration in the parasites is unusually low. 3A. Schmidt and R. L. Krauth-Siegel, unpublished observations. Therefore the trypanothione/tryparedoxin system described here is supposed to be the main donor of reducing equivalents for the parasite synthesis of deoxyribonucleotides. We thank Drs. A. Hofer and L. Thelander, Umeå, Sweden for providing us with the clones of T. bruceiribonucleotide reductase.