Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase*

Matthias DormeyerDagger, Nina Reckenfelderbäumer, Heike Lüdemann, and R. Luise Krauth-Siegel§

From the Biochemie-Zentrum Heidelberg, Universität Heidelberg, 69120 Heidelberg, Germany

Received for publication, November 15, 2000, and in revised form, December 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 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. brucei tryparedoxin 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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1). African trypanosomes possess a typical eukaryotic class I ribonucleotide reductase (2-4). The genes encoding the Trypanosoma 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 (1, 5). T. brucei ribonucleotide 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 (6).

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 (7, 8). Oxidized thioredoxin is subsequently reduced by thioredoxin reductase at the expense of NADPH (8). The dithiol form of glutaredoxin is spontaneously regenerated by glutathione. Glutathione disulfide formed in the reaction is then reduced by NADPH and glutathione reductase (9, 10).

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 and T. 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 (11, 12). These glutathionylspermidine conjugates are kept reduced by the flavoenzyme trypanothione reductase (TS2 + NADPH + H+ right-arrow T(SH)2 + NADP), an essential enzyme of the parasite (13, 14).

Trypanothione spontaneously reduces dehydroascorbate (15) and hydrogen peroxide (16). The latter reaction is catalyzed by an enzyme cascade composed of trypanothione, trypanothione reductase, tryparedoxin, and a tryparedoxin peroxidase (17, 18). Tryparedoxin is a 16-kDa protein with an active site WCPPC motif (19, 20). 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 (20).

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 for T. brucei ribonucleotide reductase and that the reaction is catalyzed by tryparedoxin.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- [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 encoding T. brucei R1 and R2 were kindly provided by Drs. Anders Hofer and Lars Thelander, Umeå, Sweden. Recombinant T. brucei tryparedoxin (20), ribonucleotide reductase (2, 3), and T. cruzi trypanothione reductase (21, 22) 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 Assay-- Ribonucleotide reductase activity was determined from the rate of conversion of [3H]GDP into [3H]dGDP essentially as described for CDP reduction (23). 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. brucei R1 (about 40 µg of protein approx  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 (24).

Ribonucleotide Reductase Activity in the Presence of Different Thiols-- 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.

Chemical Reduction of Trypanothione Disulfide-- 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. 25).

Carboxamidomethylation of Tryparedoxin-- 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 mM iodoacetamide 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 for Crithidia fasciculata tryparedoxin (19). 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 (25). 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Different Thiols as Hydrogen Donors of T. brucei Ribonucleotide Reductase-- 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. The Km 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 (26, 27) 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).



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Fig. 1.   Activity of T. brucei ribonucleotide reductase in the presence of different thiols and tryparedoxin. Formation of dGDP was followed as described under "Experimental Procedures." The assay mixtures contained 1 mM DTE, 1 mM T(SH)2, 2 mM Gspred, and 2 mM GSH, respectively (light gray columns), and additionally, 4 µM T. brucei tryparedoxin (dark gray columns). The reactions were started by adding 62 µg of R1 and 92 µg of R2 protein. The data represent a typical experiment out of five, the values of which differed by less than 10%.


                              
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Table I
Kinetic parameters of T. brucei ribonucleotide reductase
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.

Effect of Tryparedoxin on Ribonucleotide Reductase Activity-- 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 (20). 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 µM trypanothione, 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).



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Fig. 2.   Determination of the Km value of T. brucei ribonucleotide reductase for tryparedoxin. A constant concentration of 2.5 mM T(SH)2 was generated enzymatically by NADPH and trypanothione reductase. The concentration of tryparedoxin was varied between 1 and 20 µM. Km and Vmax values were derived from the Lineweaver-Burk plot (inset). The assays were performed as described under "Experimental Procedures." The activities were corrected for the spontaneous rate observed at 2.5 mM trypanothione. The data represent a typical experiment out of four that differed by less than 15%.

Alkylation of Tryparedoxin Abolishes Reduction of Ribonucleotide Reductase-- Tryparedoxin was carboxamidomethylated with iodoacetamide under nondenaturing conditions that, in analogy to E. coli thioredoxin (28) and C. fasciculata tryparedoxin (19), 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).


                              
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Table II
Effect of carboxamidomethylation of T. brucei tryparedoxin on ribonucleotide reductase activity
All reaction mixtures contained 250 µM trypanothione. The tryparedoxin concentration was 4 µM. The assays were performed as described under "Experimental Procedures."

The Activity of Tryparedoxin Is Inhibited by Trypanothione Disulfide-- The effect of TS2 on the activity of T. 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. 3a). 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 µM tryparedoxin inhibited the rate of deoxyribonucleotide formation by 90% (Fig. 3b). 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.



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Fig. 3.   Influence of TS2 on (a) the trypanothione- and (b) the T(SH)2/tryparedoxin-mediated synthesis of dGDP by T. brucei ribonucleotide reductase. The assays were performed as described under "Experimental Procedures." All reaction mixtures contained 1 mM trypanothione, 40 µg of R1 and 60 µg of R2 protein, and variable concentrations of TS2 as indicated. Columns marked by TR/NADPH represent assays that also contained 100 µM NADPH and 100 milliunits of trypanothione reductase. A typical experiment out of three is given that differed by less than 10%.

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 TS2 at the beginning, in addition to trypanothione disulfide formed during the reaction, and explains the pronounced effect of trypanothione reductase/NADPH.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (11, 13-17). 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. brucei ribonucleotide 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 (29). 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. 11). 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 (30). 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 (31). Trypanothione is the first example of a natural low molecular mass dithiol that is a direct reductant of ribonucleotide reductase.



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Fig. 4.   Scheme for the two trypanothione-dependent reactions of T. brucei ribonucleotide reductase. Left, spontaneous reduction of ribonucleotide reductase by trypanothione. Right, tryparedoxin-mediated reduction of ribonucleotide reductase by trypanothione.

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 (17-19). The apparent Km value of T. brucei ribonucleotide reductase for T. brucei tryparedoxin (3.7 µM) is higher than those of E. coli ribonucleotide reductase for glutaredoxins 1 and 3 (0.13 and 0.35 µM, respectively) (32). It is comparable with that for thioredoxin (1.3 µM) in the E. coli system (10). The maximum activity of recombinant T. brucei ribonucleotide 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 (10, 33) and thus is significantly higher than that of E. coli ribonucleotide reductase with glutaredoxin 3 as hydrogen donor where the Vmax is only 5% that of glutaredoxin 1 (32).

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 (17, 34). 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 (19, 20, 35). The trypanothione concentration in T. brucei is 400-800 µM (11), 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 (17). 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 (11) 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 of T. 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) (10) 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 (10). In contrast, with respect to the protein sequence, as well as thiol-disulfide exchange reactions, tryparedoxin is more similar to thioredoxins (20). 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 (32, 33). Recently we have cloned the gene encoding a classical thioredoxin from T. brucei (36). The recombinant protein is also a hydrogen donor for the trypanosomal ribonucleotide reductase when applied together with DTE or NADPH and human thioredoxin reductase.2 The thioredoxin gene is expressed throughout the life cycle of T. brucei, but the protein concentration in the parasites is unusually low.3 Therefore the trypanothione/tryparedoxin system described here is supposed to be the main donor of reducing equivalents for the parasite synthesis of deoxyribonucleotides.


    ACKNOWLEDGEMENTS

We thank Drs. A. Hofer and L. Thelander, Umeå, Sweden for providing us with the clones of T. brucei ribonucleotide reductase.


    FOOTNOTES

* This work is supported in part by the Deutsche Forschungsgemeinschaft (SFB 544; Kontrolle tropischer Infektionskrankheiten). M. D. was supported by a Kekulé scholarship of the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Current address: 4SC GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany.

§ To whom correspondence should be addressed: Biochemie-Zentrum Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany. Tel.: 49 6221 54 41 87; Fax: 49 6221 54 55 86; E-mail: krauth-siegel@urz.uni-heidelberg.de.

Published, JBC Papers in Press, January 9, 2001, DOI 10.1074/jbc.M010352200

2 H. Schmidt and R. L. Krauth-Siegel, unpublished results.

3 A. Schmidt and R. L. Krauth-Siegel, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: R1 and R2, large and small subunit, respectively, of ribonucleotide reductase; DTE, dithioerythritol; DTNB, 5,5'-dithiobis(2-nitrobenzoate); Gsp, (mono)glutathionylspermidine; T(SH)2, trypanothione [N1,N8-bis(glutathionyl)spermidine]; TS2, trypanothione disulfide (oxidized trypanothione); TR, trypanothione reductase; HPLC, high pressure liquid chromatography.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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