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INTRODUCTION |
Parasitic trypanosomatids are the causative agents of
life-threatening tropical diseases such as American Chagas' disease (Trypanosoma cruzi), African sleeping sickness
(T. brucei gambiense and T. b.
rhodesiense), Nagana cattle disease (T. congolense, T. b. brucei), and the three manifestations
of leishmaniasis. All these protozoa have in common that they possess
an unparalleled thiol metabolism (1-3) where glutathionylspermidine
conjugates are the main non-protein thiols. Trypanothione
(T(SH)2;
N1,N8-bis(glutathionyl)spermidine)
(4) is kept in the dithiol state by the flavoenzyme trypanothione
reductase, which catalyzes the NADPH-dependent reduction of
trypanothione disulfide. Several genetic approaches revealed that
trypanothione reductase is essential for the parasites (Ref. 5, for a
recent review see Ref. 3).
So far the trypanothione metabolism is the only known thiol system of
the parasites where it replaces the otherwise ubiquitous glutathione/glutathione reductase couple. We could show that African trypanosomes contain also a classical thioredoxin but the respective specific reductase has not yet been described in any kinetoplastid organism (6). Trypanothione is the donor of reducing equivalents in
several vital pathways of the parasites, like reduction
dehydroascorbate (7), synthesis of deoxyribonucleotides by
ribonucleotide reductase (8), conjugation and export of metals and
drugs (9-11), and the detoxication of hydroperoxides (12-14).
Trypanosomes are exposed to various reactive oxygen species such as
superoxide anions, hydrogen peroxide, and products of the host
myeloperoxidase system. However, their ability to cope with oxidative
stress seems to be surprisingly weak. Although pathogenic trypanosomes
possess an iron-containing superoxide dismutase, they lack both
catalase and a selenocysteine-containing classical glutathione
peroxidase (GPX),1 which are
the main hydroperoxide metabolising enzymes in the mammalian host. In
trypanosomatids, the metabolism of hydrogen peroxides is achieved by a
unique trypanothione-dependent cascade first described in
the insect parasite Crithidia fasciculata (12, 13). With
NADPH as final electron donor, the reducing equivalents flow from
trypanothione onto tryparedoxin and finally a tryparedoxin peroxidase,
which then catalyzes the reduction of the hydroperoxide. The latter
enzyme is a member of the 2-Cys peroxiredoxin protein family.
Components of this pathway have been demonstrated in many trypanosomatid organisms (14) such as C. fasciculata,
T. brucei, T. cruzi, Leishmania major,
and L. donovani. Distinct mitochondrial tryparedoxin
peroxidases have been found in T. cruzi (15) and T. brucei (16).
Classical glutathione peroxidases are selenoproteins, which catalyze
the reduction of different hydroperoxides at the expense of glutathione
(17-19). Four distinct selenocysteine-glutathione peroxidases occur in
mammals, cytosolic GPX 1, also called classic or cellular GPX,
gastrointestinal GPX 2, the GPX 3 found in human plasma, and the
phospholipid hydroperoxide glutathione peroxidase GPX 4 (19, 20).
Classical glutathione peroxidases have so far only been detected in
vertebrates and a GPX 4 in the helminth Schistosoma mansoni
(21). Genes encoding homologous proteins in which the active site
selenocysteine is replaced by cysteine are widely distributed in nature
but in most cases catalytic activities have not yet been demonstrated.
Recently a putative cysteine-containing glutathione peroxidase has been
studied in the malarial parasite Plasmodium falciparum. The
recombinant protein proved to be much more efficient with thioredoxin
than with glutathione as electron source (22). In T. cruzi
the gene of a cysteine homologue of glutathione peroxidases has been
cloned and overexpressed and the recombinant protein has been described
as glutathione peroxidase (23). The reported activity with glutathione
was extremely low, resulting in an apparent Vmax
of 30 milliunits/mg in the presence of 3 mM GSH and a
Km for GSH of about 7 mM. Recently the
enzyme was shown also to use trypanothione/tryparedoxin as electron
source to reduce different hydroperoxides but not hydrogenperoxide (24). The marginal activity of the T. cruzi enzyme with GSH and the fact that the GSH concentration is conspicuously low in trypanosomes prompted us to study the respective peroxidase system in
African trypanosomes and to elucidate the physiological electron donor(s). Here we report on the cloning and functional characterization of the T. brucei peroxidase. The enzyme accepts different
thiols as substrate but shows a very high preference for the
trypanothione/tryparedoxin couple.
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EXPERIMENTAL PROCEDURES |
Materials--
Human thioredoxin reductase and human glutathione
reductase were a kind gift from Dr. Heiner Schirmer, Heidelberg.
Recombinant T. brucei thioredoxin (6) and tryparedoxin (25)
and T. cruzi trypanothione reductase (26) were prepared as
described earlier. A P1 library of T. brucei strain TREU
927/4 (27) on high density filters and the positive P1 clones were
kindly provided by Dr. Vanessa Leech, Cambridge, UK. Polyclonal rabbit
antibodies against the recombinant T. brucei peroxidase were
produced by BioScience, Göttingen. Cultured procyclic T. brucei cells of strain 449 [strain 427 (MiTat 1, Ref. 28) stably
transfected with pHD 449 coding for the tetracycline repressor (29)]
and of strain 927-449 (TREU 927/4 stably transfected with pHD 449)
were kindly provided by Dr. Christine Clayton, Heidelberg. Long slender
and short stumpy cells isolated from mice, and cultured procyclic cells
(all of strain AnTat 1.1, Ref. 30) were a gift of Dr. Dietmar
Steverding, Heidelberg. Isolated T. brucei mitochondria were
kindly provided by Dr. André Schneider, Fribourg, Switzerland.
Trypanothione disulfide was purchased from Bachem, t-butyl
hydroperoxide and cumene hydroperoxide from Sigma.
PCR Amplification and Sequence Analysis of the Peroxidase
Locus--
Total RNA of T. brucei bloodstream form
parasites of strain 449 was reverse transcribed with
OmniscriptTM Reverse Transcriptase (Qiagen). The cDNA
was amplified by 10 cycles of PCR using a spliced leader primer
(5'-CTATTATTAGAACAGTTTCTGTAC-3') and an oligo(T) primer (95 °C, 2 min; 95 °C, 1 min; 50 °C, 1 min; 72 °C, 5 min; 10 cycles;
72 °C, 10 min). The 5'-region of the peroxidase gene was amplified
from the cDNA pool with a gene-specific primer
(5'-ATCAATGAGGAAGGATGTAAAGTTCC-3') and the spliced leader primer. For
the 3'-region a gene-specific primer (5'-CTCATGCTGCGTTCATCTCGG-3') and
the oligo(T) primer were used (94 °C, 2 min; 94 °C, 30 s; 50 °C, 30 s; 72 °C, 3 min; 30 cycles; 72 °C, 5 min). Two
specific primers (5'-GCGCGGATCCCTGCGTTCATCTCGG-3' and
5'-GCATGGTACCCAGCGACAAATCC-3') were derived from the
resulting sequences for gene amplification from genomic DNA of
bloodstream strain 449 (94 °C, 2 min; 94 °C, 30 s; 50 °C,
30 s; 72 °C, 2 min; 30 cycles; 72 °C, 5 min). The PCR
product was cloned using the BamHI and KpnI
restriction sites (underlined sequences) into the pQE-30 vector
(Qiagen) and used as digoxigenin-labeled hybridization probe for
screening the P1 library. Labeling and luminescence detection
components from Roche Molecular Biochemicals were used as described by
the provider. Four positive clones were isolated, digested with
PvuII and BamHI, and the three positive fragments
obtained were subcloned into the pBluescript KS(+) vector (Stratagene).
Both strands of the subclones were completely sequenced by MWG Biotech.
The sequence of the complete peroxidase locus later revealed that the
5'- and 3'-primers used to generate the hybridization probe
corresponded to px III and px I or II,
respectively, and that the genomic amplification had generated a
chimeric product. Therefore for the functional characterization of the
peroxidase III (Px III) protein, the authentic gene was cloned and
overexpressed as described below.
Preparation of Genomic DNA and Southern Blot
Analysis--
Genomic DNA of T. brucei strain 449 was
prepared using the DNeasy tissue kit (Qiagen). 10 µg of DNA was
digested with the respective restriction enzyme. The fragments were
separated on a 1% agarose gel, blotted onto HybondTM-N+
membrane (Amersham Biosciences), and hybridized with the
digoxigenin-labeled peroxidase gene.
Preparation of Poly(A)+ mRNA and Northern Blot
Analysis--
Total RNA from cultured T. brucei cells of
procyclic and bloodstream strains 449 (7 × 108 cells)
was prepared with the RNeasy Kit (Qiagen). Poly(A)+
mRNA was enriched from 475 µg of total RNA using the
Oligotex® mRNA Kit (Qiagen) and separated on a 1%
agarose gel containing 1.8% formaldehyde. Blotting and hybridization
were performed as described above.
Heterologous Expression of the Peroxidase III Gene and
Purification of the Recombinant Protein--
For the production of the
recombinant peroxidase III with an N-terminal His6 tag, the
coding region without the initial ATG was amplified by PCR using
gene-specific primers (5'-GCGCGGATCCCTGCGTTCATCTCGG-3' and
5'-GCATGGTACCGTCCAGAGACGTATTC-3') and genomic DNA from
procyclic strain AnTat 1.1 (94 °C, 2 min; 94 °C, 30 s;
50 °C, 30 s; 72 °C, 2 min; 35 cycles; 72 °C, 5 min, Pfu
polymerase). The amplification product was digested with
KpnI and BamHI, cloned into the pQE-30 vector
(Qiagen), and sequenced. Competent Escherichia coli XL-1 Blue cells were transformed with the pQE-30/px III plasmid.
A 5-ml overnight culture was diluted 1:100 in 2× YT medium containing 100 µg/ml carbenicillin. The cells were grown at 37 °C and 180 rpm
to an OD600 of 0.6. Expression was induced by adding 1 mM isopropyl-
-D-thiogalactopyranoside
overnight at 30 °C. After centrifugation, the cells were resuspended
in 15 ml of buffer A (50 mM sodium phosphate, 300 mM NaCl, pH 8.0) containing 10 mM imidazole,
150 nM pepstatin, 4 nM cystatin, 100 µM phenylmethylsulfonyl fluoride, 1.5 mg lysozyme, 0.15 mg DNase A, and disintegrated by sonification. Following
centrifugation, the supernatant was applied onto a 4-ml
nickel-nitrilotriacetate matrix Superflow column (Qiagen). The column
was washed with 50 ml of 25 mM imidazole in buffer A, and
the His-tagged protein was eluted with 250 mM imidazole in
buffer A. The protein fractions were analyzed for homogeneity by
SDS-polyacrylamide gel electrophoresis and stored at 4 °C. The
protein concentration was determined using the bicin choninic acid kit
(Pierce). For homogenous peroxidase III, a protein concentration of 1 mg/ml corresponds to an
A280 of 1.
Gel Filtration--
The subunit composition of T. brucei peroxidase III was determined by FPLC on a Superose 12 HR
10/30 column (Amersham Biosciences). 160 µg of purified recombinant
protein was applied onto the column equilibrated in 0.1 M
potassium phosphate buffer, 1 mM EDTA, pH 7.0 and run at
room temperature with a flow rate of 0.2 ml/min. Bovine serum albumin,
rabbit aldolase, horse cytochrome c, and ferritin served as
molecular mass standards.
Cultivation of Bloodstream T. brucei--
Culture-adapted
bloodstream T. brucei of cell line 449 were grown in HMI-9
medium (31) supplemented with 1.5 mM cysteine, 0.0014%
(v/v) 2-mercaptoethanol, 10% heat-inactivated fetal calf serum, and
0.2 µg/ml phleomycin at 37 °C in a humidified atmosphere with 5%
CO2.
Western Blot Analysis--
Cultured cells and mice isolates
(2 × 106 trypanosomes each) were centrifuged. The
cell pellets were mixed with 20 µl of loading buffer (0.25 mM Tris, pH 6.8, 8% (v/v) SDS, 40% (v/v) glycerol, 50 mM DTE, 0.04% (w/v) bromphenol blue) and directly loaded
onto a 15% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred onto a HybondTM-P membrane (Amersham
Biosciences) by semidry electroblotting and probed with the polyclonal
rabbit antiserum against the recombinant protein (dilution 1:1000).
Anti-rabbit IgG (dilution 1:5000) served as secondary antibody. The
immune complex was visualized using the ECL plus system (Amersham Biosciences).
Activity Measurements--
Glutathione peroxidase activity was
measured at 25 °C in a total volume of 90 µl in 0.1 M
Tris, 5 mM EDTA, pH 7.6, containing 240 µM
NADPH, 2.5-12 mM GSH, 75 milliunits of human glutathione reductase, 4 µM T. brucei peroxidase, and 50 µM hydroperoxide. The reaction was followed at 340 nm by
measuring the glutathione reductase catalyzed oxidation of NADPH. The
total concentration of the hydroperoxide substrate was determined by
allowing the reaction to run to completion, and the concentration at
each time point was calculated from the initial concentration. The
assays were standardized with glutathione peroxidase from bovine
erythrocytes (Sigma). Thioredoxin peroxidase activity was assayed as
described above except that GSH and glutathione reductase were replaced by 1.5-20 µM T. brucei thioredoxin
and human thioredoxin reductase. The
trypanothione/tryparedoxin-dependent peroxidase activity was measured at a fixed concentration of 100 µM
trypanothione, 90 milliunits of trypanothione reductase, 0.6-8.3
µM tryparedoxin, and 1 µM peroxidase. All
other parameters were those described above for the glutathione
peroxidase assay. The reactions were started by adding the
hydroperoxide substrate (H2O2,
t-butyl hydroperoxide, cumene hydroperoxide). Control assays
lacked the peroxidase, tryparedoxin, GSH, thioredoxin, and
trypanothione, respectively.
Kinetic Analysis of the Peroxidase--
Concentration dependence
of the initial velocities was obtained by analyzing the time
progression curve of NADPH consumption in a Beckman DU 65 spectrophotometer with data acquisition every 1.5 s. The data were
analyzed using the integrated Dalziel equation for a two substrate
enzyme reaction (32, 33) shown in Equation 1,
|
(Eq. 1)
|
where [ROOH] is the initial hydroperoxide concentration,
[ROH]t the concentration of the product at
time t, [Thiol] the concentration of the respective thiol
substrate, [E]0 the total concentration of the peroxidase
and
0,
1, and
2 are the kinetic Dalziel coefficients.
 |
RESULTS |
Analysis of the Genomic Locus of the Trypanosoma brucei
Peroxidase--
The genome sequencing project of Trypanosoma
brucei revealed several fragments encoding a glutathione
peroxidase-related gene. Based on this information, the gene has been
cloned from cDNA of bloodstream parasites of strain 449. The
5'-region was amplified by PCR using a sequence specific reverse primer
and a spliced leader primer, which is the very 5'-end of all
trypanosomal mRNAs added by transsplicing. The 3'-region was
amplified with a specific forward primer and a poly(T) primer. The
complete coding region of the gene was then amplified from genomic DNA
with two gene-specific primers and used as hybridization probe in
Southern blot analysis. Digestion of T. brucei genomic DNA
(procyclic strain 927-449) with several restriction enzymes revealed
the presence of more than one peroxidase gene (Fig.
1A). The combined digestion
with PvuII and BamHI resulted in three fragments
of
0.9 kb, each fragment being larger than the coding region of the
gene (about 0.5 kb) used for hybridization. The upper band in the
PvuII digest represents two fragments of which one is
cleaved in the double digestion with BamHI yielding the
1.2-kb fragment. To elucidate the complete structure of the locus, a P1
library of T. brucei genomic DNA (strain TREU 927/4) on high
density filters was screened with the peroxidase probe. Four positive
clones were obtained which yielded an identical restriction pattern
(data not shown). The three positive fragments of P1 clone 21E1 were
cloned and sequenced (EMBL/GenBankTM accession number
AJ298281). The sequence and Southern blot data were in accordance with
a genomic locus of 4.4 kb containing three clustered peroxidase genes
(px I-px III) (Fig. 1B). The existence of more than three
gene copies can be excluded since the BsaI/BamHI
fragment of 3.2 kb comprises all three peroxidase genes. Further
thorough Southern blot analyses of genomic DNA digested with
EcoRI, EcoRV, BamHI,
HindIII, Bsp143I, and BsaI fully
agreed with the arrangement of three genes depicted in Fig. 1B. Some of the blots showed a few very faint additional
bands, which may indicate the existence of distantly related gene(s) outside this locus.

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Fig. 1.
Southern blot analysis and restriction map of
the peroxidase locus of T. brucei. A, digested
DNA of strain TREU 927/4 was separated on a 1% agarose gel. The blot
was hybridized with a DIG-labeled PCR-probe of the coding region as
described under "Experimental Procedures." B, the scheme
gives the restriction sites and the arrangement of the three peroxidase
genes. The arrow indicates the direction of
transcription.
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Structural Comparison of the Three T. brucei Thiol
Peroxidases--
The three protein sequences deduced from the genomic
peroxidase locus consist of 166, 169, and 176 amino acid residues,
respectively (Fig. 2). The overall
sequences are almost identical except for the very N- and C-terminal
regions. Peroxidase III, which is characterized here, is the largest
one and has a calculated pI value of 9.5. In contrast, the peroxidases
I and II have pI values of 6.28 and 8.34, respectively. The very basic
pI of peroxidase III is mainly due to its N-terminal extension. The
sequence MLRSSRKKMSAA is rich in positively charged and hydroxylated
residues and shows homology to other exceptionally short (7-9 amino
acid residues) mitochondrial targeting signals of trypanosomatids (34,
35). When the gene is overexpressed from the pQE-30/pxIII in
E. coli, two protein species are obtained, one with the
His6 tag and the complete N-terminal sequence and a second
about a 2-kDa smaller protein, which does not bind to the metal matrix.
Both proteins are fully active. N-terminal Edman degradation revealed
that the recombinant protein has been cleaved between two basic
residues of the RKK motif resulting in a protein starting with
(K)KMSAA. The motif is obviously recognized by a bacterial protease.
Interestingly, T. brucei possesses an endopeptidase which
preferentially cleaves after two basic residues and which has been
discussed to play a role in protein processing (36). Px III has also a
C-terminal ARL motif, which could be a variation of the peroxisomal and
glycosomal PTS 1 targeting signal SKL. Since such variations have been
shown to be functional (37), the peroxidase III might be also localized in glycosomes. The structurally closely related T. cruzi
peroxidase I possesses a C-terminal ARI sequence and occurs both in the
cytosol and in glycosomes (24). The other two T. brucei
proteins Px I and Px II lack known signal peptides and are probably
located in the cytoplasm of the parasites. Except from the very N- and C-terminal regions, only eight residues are not identical in the three
proteins (Fig. 2). Five substitutions are found only in Px I and the
other three in the Px II sequence. In any case, only two different
residues occur at the respective position. Two of these biases
(positions 59 and 86 in Px III) are even present when comparing the
peroxidase III sequences from strain TREU 927/4 (27) and strain AnTat
1.1 (30). With the possible exception of lysines 131 and 165, which
occur only in the peroxidase II, the substitutions seem to be randomly
distributed in accordance with a microheterogeneity of these genes.
Taken together, the data suggest very similar or identical catalytic
properties for the three peroxidases.

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Fig. 2.
Alignment of the three T. brucei
peroxidases I, II, and III from strain TREU 927/4 and peroxidase
III (A1.1) from strain AnTat 1.1. The non-identical residues at
the N and C termini are given in italic letters, varying
internal residues are depicted in bold and
italics.
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Structural Comparison of the T. brucei Peroxidases with Classical
Glutathione Peroxidases--
The primary structures of the T. brucei peroxidases described here classify the proteins as members
of a large protein family comprising the selenocysteine-containing
glutathione peroxidases (GPX). Classical glutathione peroxidases have a
strictly conserved catalytic triad composed of a Se-Cys, a Gln, and a
Trp residue (Fig. 3). The highest
similarity of the T. brucei peroxidases is found with a
T. cruzi protein described as glutathione peroxidase I (23,
24) where 72% of all residues are conserved (Fig. 3). In the
trypanosomal proteins the selenocysteine is replaced by a cysteine
residue as it is the case in the protein of the malarial parasite
P. falciparum (22) and many functionally not well
characterized plant proteins. To other members of the family the
highest degree of similarity is found with glutathione peroxidase-like
proteins from plants; for instance 49% of all residues are identical
in the putative phospholipid hydroperoxide glutathione peroxidase from
Arabidopsis thaliana (38). The closest relative of the four
human selenocysteine-containing glutathione peroxidases is the
phospholipid hydroperoxide GPX 4 (39% identity), followed by the
cytosolic GPX 1 (35%), the gastrointestinal GPX 2 (30%), and the
extracellular GPX 3 (25%).

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Fig. 3.
Comparison of the T. brucei
peroxidase III with glutathione peroxidases from other
organisms. The catalytic triad is marked by triangles.
U represents selenocysteine. Residues identical in at least
seven of the nine sequences are depicted in bold letters.
T. cruzi I, T. cruzi glutathione peroxidase I
(23), acc. no. CAC85914; A. thaliana PL, putative
phospholipide hydroperoxide glutathione peroxidase from A. thaliana (acc. no. BAA24226); P. falciparum, P. falciparum thioredoxin peroxidase (acc. no. CAA92396); S. mansoni, S. mansoni (acc. no. Q00277). The four human
enzymes are: 1) cytosolic or cellular glutathione peroxidase (GPX 1)
(acc. no. P07203); 2) gastrointestinal (GPX 2) glutathione peroxidase
(acc. no. P18283); 3) plasma glutathione peroxidase (GPX 3) (acc. no.
P22352); and 4) phospholipid hydroperoxide glutathione peroxidase (GPX
4) (acc. no. CAA 50793).
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The 3-dimensional structures of bovine erythrocyte glutathione
peroxidase (GPX 1) and of the human plasma glutathione peroxidase (GPX
3) (39, 40) have been solved in free form and the residues responsible
for substrate binding have not yet been elucidated. For the cytosolic
bovine GPX 1, several basic residues have been suggested to play a role
in glutathione binding (39-41). None of the residues is conserved in
the T. brucei peroxidases and only Arg-103 in the
extracellular human enzyme. If the residues are indeed involved in
glutathione binding this finding is in accordance with the latter two
enzymes not using glutathione as preferred thiol substrate (Ref. 42 and
see below).
The Peroxidase Is Expressed in All Developmental Stages of
Trypanosoma brucei--
Northern blot analysis of poly(A)+
RNA from cultured procyclic and bloodstream parasites revealed a single
mRNA of about 1 kb in both stages (data not shown). For Western
blot analysis, cell lysates of cultured bloodstream and procyclic
parasites as well as of long slender and short stumpy forms isolated
from mice were prepared. The polyclonal rabbit antibody against the
recombinant peroxidase detected a protein band in all stages in
accordance with a general function of the peroxidase(s) throughout the
life cycle of T. brucei (Fig.
4). Due to the high sequence homology of
the three peroxidases, it is not possible to distinguish the expression
of the individual proteins. Western blot analysis of isolated
mitochondria from procyclic T. brucei revealed a
corresponding single protein band (data not shown), which confirms that
the very short N-terminal stretch of Px III is indeed a mitochondrial import signal.

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Fig. 4.
Western blot analysis of the peroxidase in
different developmental stages of T. brucei.
Extracts of about 2 × 106 parasites and 10 ng of
recombinant peroxidase were subjected to PAGE on a 15% SDS gel.
Polyclonal rabbit antibodies against the recombinant protein together
with an ECL detection kit were used for visualization. ls,
long slender and ss, short stumpy bloodstream parasites from
mice; pc, procyclic and bf, bloodstream culture
form; His-Px, recombinant His-tagged peroxidase III.
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The Recombinant Peroxidase III Is a Monomer--
The
pxIII gene was expressed from the pQE-30/px III
vector in E. coli XL-1 blue cells. Purification of the
N-terminally His6-tagged protein on a nickel ligand column
yielded about 15 mg of pure recombinant protein from 1 liter of
bacterial culture. On SDS-PAGE, the recombinant peroxidase showed a
molecular mass of 21,000, 2 kDa larger than the authentic
protein (Fig. 4). Gel filtration on Superose 12 revealed an
Mr of about 20,000 for the recombinant His-tagged protein in accordance with the enzyme being a monomer as it
is the case for porcine phospholipid hydroperoxide GPX 4 (43). Both
proteins lack a stretch of about 15 residues (following Thr-132), which
has been shown to be a subunit interaction site in the tetrameric GPXs
(39).
Different Thiol Systems as Electron Donors for the T. brucei
Peroxidase--
T. brucei peroxidase III catalyzes the
reduction of hydrogen peroxide and organic hydroperoxides in the
presence of different thiol substrates. Glutathione, T. brucei thioredoxin, and the trypanothione/T. brucei
tryparedoxin couple were studied as electron donors. The kinetic
mechanism of the parasite enzyme was evaluated by single curve
progression analysis (32, 33) at several fixed thiol concentrations.
The hydroperoxide concentration was chosen such that the reaction rate
was dependent on its concentration. The thiol concentration was kept
constant over time by coupling the reaction to the respective
reductase, which allowed the reactions to be followed by measuring
NADPH consumption. The reciprocal initial velocities multiplied by the
enzyme concentration were plotted against the reciprocal concentrations
of the hydroperoxide substrate. These plots yielded parallel lines
(Fig. 5A), which implies an
enzyme substitution or ping-pong mechanism as described by the special
Dalziel equation for bisubstrate reactions (32) as shown in Equation 2,

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Fig. 5.
Kinetic analysis of the peroxidase catalyzed
reduction of cumene hydroperoxide in the presence of tryparedoxin.
A, linear plot of the integrated Dalziel rate equation. The
activities were measured at a fixed concentration of 100 µM trypanothione and five different concentrations of
tryparedoxin: 0.6 µM 1 ( ), 1.1 µM 1 ( ), 2.5 µM 1 ( ), 5 µM 1 ( ), and 8.3 µM 1 (+). The hydroperoxide concentration at
each time point was obtained by single curve progression analysis
according to Forstrom et al. (33). The ordinate
[E]0 × t/([S0] [S]) represents
[E]0/v, and the abscissa is equivalent to 1/[ROOH]. The
empirical Dalziel coefficient 1 is given by the slope of
the linear regressions. B, secondary Dalziel plot. The
apparent maximum velocities for infinite hydroperoxide concentrations
obtained from Fig. 5 were plotted against the reciprocal concentrations
of tryparedoxin. The slope represents the Dalziel coefficient
2, and the ordinate intercept corresponds to
0. Intersecting the ordinate at or near zero implies
that maximum velocity and Km values of the enzyme
are infinite for the respective substrate pair.
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(Eq. 2)
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where ternary complexes between the enzyme and both substrates are
not formed. In this equation
0 defines
[E]/Vmax, the reciprocal enzyme-normalized
limiting maximum turnover;
1 and
2 are
k1' and k2',
respectively, the overall reciprocal rate constants of the two half
reactions (Fig. 6). Most probably the peroxidase reacts with its substrates in two independent consecutive reactions, oxidation of the reduced peroxidase by ROOH, and subsequent reduction of the oxidized enzyme by the respective thiol. An analogous kinetic has been found and analyzed in detail in the case of the peroxiredoxin-like peroxidases in trypanosomatids (12, 13). A replot of
the ordinate intercepts, which represent the reciprocal apparent
maximum velocities at infinite peroxide concentration, against the
reciprocal thiol concentration, yields a straight line that cuts the
ordinate at
0. As outlined below, with thioredoxin and
the trypanothione/tryparedoxin couple as thiol donors, the T. brucei peroxidase yielded secondary Dalziel plots intersecting the
ordinate at or near zero (Fig. 5B and Table
I). The fact that the term
0 approaches zero implies that the kinetic does not show
typical saturation at high concentrations of both substrates but that
the limiting Vmax and Km
values are infinite. The kinetic pattern does not exclude formation of
a specific enzyme-substrate complex, but if it is formed its decay
cannot be the rate-limiting step in the overall reaction (Fig. 6).

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Fig. 6.
The two partial reactions catalyzed by the
peroxidase. k1' is
k+1 k-1 and may be
regarded as k+1 since the partial reaction
should be irreversible. k2' is the overall rate
constant for the two-step regeneration of reduced enzyme by the thiol.
The reciprocal rate constant k1' is defined as
1, whereas the reciprocal k2' is
given by 2.
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Table I
Kinetic coefficients and apparent rate constants of T. brucei
peroxidase III for the reduction of hydroperoxides by different thiol
substrates
The Dalziel coefficients 1 and 2 for the
thioredoxin- and tryparedoxin systems are the mean of at least three
independent series. They correspond to the reciprocal of the apparent
rate constants k1' and
k2' for the two half reactions. The extrapolated
0 coefficients were zero within the experimental error for
these reactions. The 1 coefficients for the GSH system were
determined for five different GSH concentrations. Limiting
Km values for the hydroperoxide and thiol substrate,
given as 1/ 0 and 2/ 0,
respectively, were only obtained in the glutathione system. The
Km values for cumeneOOH and t-butylOOH
were 12 and 47 µM, the Km values for
GSH were 16 and 46 mM, respectively.
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The activity of the Px III was first measured in the presence of
GSH/NADPH/human glutathione reductase with cumene hydroperoxide and
t-butylhydroperoxide as substrates. GSH concentrations
between 2.5 and 12 mM were necessary to detect enzymatic
activity at all. The primary Dalziel plots at several fixed GSH
concentrations showed linear dependence of the reciprocal enzyme
activity on the hydroperoxide concentration. Parallel lines were
obtained for the different GSH concentrations, in accordance with
ternary complexes between the enzyme and the substrates not being
formed. The replot of the ordinate intercepts versus the
reciprocal GSH concentration yielded lines that intersected the
ordinate at distinct
0 values. Taking into account the
large experimental error when measuring such low activities,
Km values of 12 and 47 µM were
obtained for cumene hydroperoxide and t-butyl hydroperoxide, respectively (Table I). The calculated Km value for glutathione was 16 and 46 mM in the presence of the two
hydroperoxides. Since the concentration of free GSH is about 134 and
300 µM in bloodstream and procyclic Trypanosoma
brucei, respectively (1), glutathione should not play a major role
as electron donor for the enzyme and the reactions were not evaluated
in further detail.
We then studied T. brucei thioredoxin and the
trypanothione/T. brucei tryparedoxin couple for their
ability to serve as electron donors of peroxidase III. The assays
contained T. brucei thioredoxin, NADPH, human thioredoxin
reductase and trypanothione, NADPH, T. cruzi trypanothione
reductase, T. brucei tryparedoxin, respectively. At
millimolar concentrations of trypanothione, direct reduction of
hydroperoxides by the dithiol becomes significant. Therefore the assays
with tryparedoxin contained a fixed concentration of 100 µM T(SH)2 where the spontaneous reaction can
be neglected. Assays lacking the peroxidase showed that tryparedoxin
alone does not accelerate the spontaneous reaction between
T(SH)2 and the hydroperoxide. A second control assay
containing all components except tryparedoxin confirmed that
trypanothione does not directly reduce the peroxidase. Thioredoxin as
well as T(SH)2/tryparedoxin were efficient electron donors
for the peroxidase (Table I). Within experimental error, the
0 coefficients could be regarded as zero, so
Vmax and Km values were
infinite for both thiol systems. Presuming a ping-pong mechanism for
the reaction, the k1' values obtained for a
single hydroperoxide substrate should be independent of the nature of
the thiol. As shown in Table I, the k1' values
varied significantly especially with GSH, which may be due to the
extremely low activities in the latter system. It is therefore not
possible to give an exact judgement of the specificity of the enzyme
toward different hydroperoxide substrates. Cumene-OOH and
H2O2 appear to be slightly better substrates
than t-butyl-OOH.
In contrast to the k1' values, which are
specific for the hydroperoxide substrate, the
k2' values represent the apparent overall rate
constants for the regeneration of the reduced enzyme and therefore
depend on the thiol. As expected, the k2' values
with different hydroperoxides are in fact almost identical for the particular thiol system. With thioredoxin a k2'
value of 5 × 103 M
1 s
1 is obtained, while the k2' value
in the T(SH)2/Tpx system is about 2 × 105
M
1 s
1. These results clearly
show that the trypanothione/tryparedoxin couple is by two and five
orders of magnitude more efficient as electron donor when compared with
thioredoxin and glutathione, respectively. Thus the enzyme, although a
structural homologue of the classical glutathione peroxidases, is
highly specific for the trypanothione/tryparedoxin couple and
represents a new class of tryparedoxin peroxidases (Fig.
7).

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Fig. 7.
Reduction of hydroperoxides (ROOH) by the
trypanothione cascade. Trypanothione reductase catalyzes the
NADPH-dependent reduction of trypanothione disulfide
(TS2) to trypanothione [T(SH)2].
Trypanothione subsequently reduces tryparedoxin (Tpxox) and
the reduced tryparedoxin (Tpxred) then transfers electrons
onto the oxidized peroxidase (Pxox). The reduced
Pxred finally catalyzes the reduction of the hydroperoxide
substrate.
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DISCUSSION |
The T. brucei peroxidase studied here is a non-selenium
homologue of glutathione peroxidases. The parasite enzyme has only marginal activity with glutathione as electron donor but shows strong
preference for T. brucei thioredoxin and especially for the
trypanothione/T. brucei tryparedoxin couple. Other proteins, which structurally belong to the glutathione peroxidase family but
prefer thioredoxin as reducing substrate, are the selenoenzyme human
plasma glutathione peroxidase (GPX 3) (42) and the non-selenium P. falciparum thioredoxin peroxidase (22). In the presence
of thioredoxin or the trypanothione/tryparedoxin couple, the T. brucei peroxidase does not show saturation kinetics toward
different hydroperoxide substrates. It follows ping-pong kinetics with
infinite Km and Vmax values
as it is the case with the classical glutathione peroxidases (17) as
well as the peroxiredoxin-like peroxidase from C. fasciculata (13). In contrast, with glutathione as thiol
substrate, saturation kinetics are observed with a
Km value for GSH of about 30 mM and
negligible kcat values of 0.05-0.1 s
1. The shift toward saturation kinetics implies that one
or more rate constants describing the decay of an enzyme-substrate
complex are slowed down when glutathione acts as the electron donor.
With glutathione as well as with thioredoxin, regeneration of the
reduced enzyme species is the rate-limiting half reaction. In contrast, in the presence of the trypanothione/tryparedoxin couple, the reaction
is much faster and reduction of the hydroperoxide substrate becomes
limiting. A similar observation has been made for the P. falciparum thioredoxin peroxidase mentioned above, where also with
glutathione regeneration of the reduced enzyme species is the slower
half reaction, whereas in the presence of thioredoxin, reduction of the
peroxide substrate is rate-limiting (22).
The closest known relative of the new T. brucei peroxidase
is a T. cruzi protein described as glutathione peroxidase I
(GPX I) (34). Despite the structural similarity (the proteins share 72% of all residues) the catalytic properties are quite different. In
contrast to the T. brucei peroxidase, the T. cruzi enzyme does not accept H2O2 as
substrate (23, 24). The marginal activity of the T. brucei
peroxidase in the presence of glutathione is comparable with that of
the T. cruzi peroxidase (23) for which an apparent
Vmax of 33 nmol/min·mg protein (corresponding
to a kcat of 0.01 s
1) has been
obtained at 3 mM GSH. Taking into account the extremely low
turnover of this system and the fact that the glutathione concentration
in trypanosomatids is in the 100-300 µM range (1) this
activity should not be of physiological significance. Recently the
T. cruzi enzyme has been shown to be reduced also by the
trypanothione/tryparedoxin couple but the activities were only 8-15
times higher than those observed with glutathione (24). The low
activities can at least partially be explained by the fact that a lower
T(SH)2 concentration was used to keep tryparedoxin reduced.
In addition, the activities refer to the overall reaction, which is
limited by the reaction between reduced trypanothione and tryparedoxin.
The T. cruzi peroxidase contains a C-terminal tripeptide ARI
and occurs both in glycosomes and in the cytosol (24). The T. brucei Px III studied here possesses a C-terminal
ARL which indicates also a glycosomal localization. In addition, its
N-terminal MLRSSRKKM sequence targets the protein into mitochondria.
Thus, the glutathione peroxidase-related peroxidase as well as the
peroxiredoxin-like enzyme (16) occur in the mitochondria of T. brucei. The first peroxidase system discovered in trypanosomatids
was the cascade composed of trypanothione, trypanothione reductase,
tryparedoxin, and the peroxiredoxin-type tryparedoxin peroxidase (13,
15). T. brucei Px III has very similar kinetic properties
and can replace the peroxiredoxin-like peroxidase in this cascade. Both
peroxidases reduce their peroxide substrates at rates of
104-105 M
1
s
1, which is slower than the subsequent regeneration of
the reduced enzyme species. In contrast, in the mammalian selenoenzyme
GPX I reduction of the peroxide substrate is extremely fast and the reductive step by glutathione which occurs at a rate of
105 M
1 s
1 becomes
limiting (17-19). Thus on a molecular basis, the parasite peroxidases
are only 5-10 times less efficient than the classical selenoenzymes.
The overall poor performance of trypanosomatids in coping with
oxidative stress can neither be attributed to the ability of the
peroxidases to reduce hydroperoxides nor to their regeneration by
reduced tryparedoxin. The limiting factor is the regeneration of
reduced tryparedoxin by trypanothione (13). As shown for different
C. fasciculata tryparedoxins, reduction of tryparedoxin
by trypanothione is much slower than the subsequent reduction of the
peroxidase by the dithiol protein, which results in a turnover number
for the overall reaction of about 5-15 s
1 (12, 44).
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CONCLUSIONS |
The identification of the new peroxidases demonstrates that two
different hydroperoxide metabolizing systems occur in T. brucei, both based on the unique trypanothione metabolism of the
parasites. The finding that the T. brucei peroxidase shows
preference for the trypanothione/tryparedoxin couple is not surprising
since only for the cytosolic classical GPX 1 a high specificity
for GSH has been demonstrated (45). In addition, it underlines the difficulty to deduce the catalytic properties of an enzyme solely from
its primary structure. The relatively weak performance of both types of
trypanothione-dependent peroxidase systems is in accordance
with the sensitivity of trypanosomes toward oxidative stress. In
addition, it cannot be ruled out that the parasite peroxidases fulfill
other tasks completely distinct from the defense against oxidative
stress. Even within the classical selenocysteine peroxidases, only GPX
1 unambiguously functions as an antioxidant enzyme, the others have
been suggested to play different roles (19). Work is in progress to
silence the peroxidase genes in bloodstream T. brucei by RNA
interference to get a deeper insight in the physiological function(s)
of these proteins.