From the Biochemie-Zentrum Heidelberg, Universität Heidelberg, 69120 Heidelberg, Germany
Received for publication, November 15, 2000, and in revised form, December 15, 2000
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ABSTRACT |
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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.
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 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+ 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.
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 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.
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).
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).
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).
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.
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.
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 (
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
T(SH)2 + NADP), an essential enzyme of the parasite (13, 14).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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%.
Kinetic parameters of T. brucei ribonucleotide reductase
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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%.
Effect of carboxamidomethylation of T. brucei tryparedoxin on
ribonucleotide reductase activity
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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%.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Hofer and L. Thelander, Umeå, Sweden for providing us with the clones of T. brucei ribonucleotide reductase.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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.
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