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INTRODUCTION |
Tryparedoxin peroxidase has recently been identified as a
constituent of the complex peroxidase system in the trypanosomatid Crithidia fasiculata (1). In these parasitic protozoa
hydroperoxides are reduced at the expense of NADPH by means of a
cascade of three oxidoreductases: the flavoprotein trypanothione
reductase, the thioredoxin-related tryparedoxin, and tryparedoxin
peroxidase (Fig. 1). The first enzyme of
the cascade is homologous to glutathione reductase and thioredoxin
reductase (2), which are involved in NADPH-dependent
hydroperoxide reduction in other species (3). The other components of
the trypanosomatid system also belong to protein families occasionally
constituting peroxidase systems. Preliminary amino acid sequencing data
indicated that tryparedoxin is phylogenetically related to thioredoxin
(1), whereas the tryparedoxin peroxidase belongs to the peroxiredoxins
(1) comprising the thioredoxin peroxidases of yeast and mammals (4) and
the alkyl hydroperoxide reductases of bacteria (5).

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Fig. 1.
Flux of reducing equivalents from NADPH to
hydroperoxide in C. fasciculata. TR,
trypanothione reductase; T(SH)2, trypanothione; TS2, trypanothione disulfide; TXN,
tryparedoxin; TXNPx, tryparedoxin peroxidase;
ROOH, hydroperoxide.
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The unique feature of the trypanosomatidal peroxidase system is its
dependence on the peculiar redox mediator trypanothione which so far
has not been discovered in any species outside the Trypanosomatidae. Its biosynthesis from spermidine and
glutathione requires two distinct enzymes, glutathionylspermidine
synthetase (6) and trypanothione synthetase (7). With a cascade of oxidoreductases plus the redox mediator trypanothione and the two
auxiliary enzymes for its synthesis, the trypanosomatids have developed
the most complicated system for the removal of hydroperoxides so far
discovered in nature. This is not to imply a particular efficiency or
robustness of the system. On the contrary, trypanosomatids are reported
to be highly susceptible to oxidative stress (8). Correspondingly,
their extraordinary metabolism is being discussed as a potential target
area of specific trypanocidal agents (1, 9, 10).
The present possibilities available for the treatment of trypanosomal
diseases, such as Chagas disease, African sleeping sickness, and the
various forms of leishmaniasis, necessitate improvement. We (1, 6),
like many others (11-14), have therefore embarked on the
identification and characterization of potential molecular targets
typical of the trypanosomatids. Here we report for the first time the
full-length DNA, deduced amino acid sequence, and expression of a
tryparedoxin peroxidase and its relatedness to peroxiredoxins with
established or unknown functions.
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EXPERIMENTAL PROCEDURES |
Cell Culture and DNA Extraction--
C. fasciculata
(HS6) was grown as described by Shim and Fairlamb (15). The cells were
harvested by centrifugation for 15 min at 7000 rpm, washed twice with
saline solution (0.9% NaCl), and resuspended in 5 ml of buffer (50 mM Tris-HCl, 100 mM EDTA, 15 mM
NaCl, 0.5% SDS, 100 µg ml
1 proteinase K, pH 8.0).
Resuspended cells were preincubated at 50 °C for 40 min. The genomic
DNA was extracted twice with equivalent volumes of phenol (incubation:
60 °C for 45 min; centrifugation: 20 min, 4500 rpm) followed by
phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl
alcohol extraction (24:1). Genomic DNA was precipitated with sodium
acetate and ethanol.
Primers, Hybridization Probes, and Sequence Analysis--
Based
upon peptide sequences of tryparedoxin peroxidase (1), degenerate
oligodeoxyribonucleotides 5'-TCGAATTCGAYATGGCSCTIATGC-3' and
5'-CTGGATCCCRATIGGCATRTC-3' were synthesized.
PCR1 amplification was
performed with the GeneAmp PCR Core kit (Perkin Elmer) using 0.8 µg
of C. fasciculata genomic DNA as template, 10 µl of
10 × reaction buffer, 8 µl of 25 mM
MgCl2, 2 µl of each 10 µM dNTP, 100 pmol of
each primer, and 0.5 unit of Taq polymerase. An annealing
temperature of 50 °C was used. The PCR product was analyzed by
agarose gels and purified using the QIAquick PCR purification kit
(QIAGEN). Sequencing was performed on a 373A DNA Sequencer (Applied
Biosystems) using the PRISM Ready Reaction DyeDeoxy Terminator Sequencing kit (1550 V, 19 mA, 30 W, 42 °C). When used as a
hybridization probe, the PCR product was labeled with digoxigenin using
the DIG DNA Labeling kit (Boehringer Mannheim) according to the
instructions provided by the supplier.
Library Construction and Screening Procedure--
The genomic
DNA was partially digested for 5-30 min with a ratio unit of
Sau3A/µg of DNA of 0.005. The efficiency of the digestion was monitored by agarose gel electrophoresis. Proteins were removed from the DNA using StrataClean Resin (Stratagene). The Sau3A
sites were partially refilled with dATP and dGTP and Klenow fragment. The genomic DNA was ligated into Lambda GEM-11 XhoI
half-site arms (Promega) at a molar ratio of
DNA to genomic DNA
(average size 15 kb) of 1:0.7. The ligated DNA was packaged using the
Packagene Lambda DNA Packaging System (Promega) according to the
suppliers' instructions. The phages were used to infect
Escherichia coli host strain LE392 (Promega) according to
the standard protocol. 5.1 × 103 plaque forming units
of the genomic library gave positive PCR signals for tryparedoxin
peroxidase and were plated on agar. The plaques were transferred to
9-cm diameter Biodyne-A nylon membranes and screened with the
digoxigenin-labeled PCR probes following the instructions provided by
the supplier but using a hybridization temperature of 58 °C.
Digoxigenin-labeled nucleic acids were detected colorimetrically with
the DIG Nucleic Acid Detection kit (Boehringer Mannheim). Positive
clones were rescreened, amplified, and suspended in SM buffer. The
phages were precipitated by PEG 8000 and purified in CsCl gradients.
The isolated DNA was used for restriction analyses (SacI) or
as template for PCR reactions. Southern blot was performed using
standard techniques, using the same digoxigenin-labeled PCR probe and
the same hybridization conditions as above. The digestion products were
eluted from agarose gels and ligated into pBluescript II KS(+/
)
phagemids (Stratagene). The ligated DNA was used to transform E. coli LE392. Transformed cells were selected by ampicillin
resistance, plasmids were purified using the QIAprep Spin Plasmid kit
(QIAGEN) and analyzed by restriction enzyme digestion and
sequencing.
Recombinant Tryparedoxin Peroxidase Expression and
Purification--
The tryparedoxin peroxidase gene contained in the
cloned 1.5-kb fragment was amplified by PCR with a forward primer A
(5'-CCACCACTTGGCGCACATATGTCCTGCGGTGCCGCC-3') that contains an
NdeI site and overlaps the 5' end of the coding sequence,
and a reverse deletion primer a
(5'-CGCGGGGTGGTTCAACTTGGCGGCACCGCAGGAC-3') to delete an extra
cytosine base at position 30. Amplification was also performed with a
forward deletion primer b (5'-CAAGTTGAACCACCCCGCGCCTGAGTTCGACGAC-3') and a reverse primer B
(5'-GCCACGCCTGCTTCTCTCCTCGAGGCCCTCCTTCTTCTTGG-3') which overlaps the 3'
end of the coding sequence and contains an XhoI site.
Consequently the last coded amino acid was changed to a leucine, a
glutamate residue was added, the stop codon was deleted, and the
protein contained 6 histidine residues at its C-terminal end.
Amplification was performed as above but using the Expand High Fidelity
polymerase mixture and buffer (Boehringer Mannheim) at an annealing
temperature of 58 °C with the extension temperature being increased
in 10-s increments per cycle during cycles 10-20. PCR products of the
expected size were obtained and used as template for a second PCR
amplification with the forward primer A and the reverse primer B. The
amplified coding region was digested with NdeI and
XhoI and ligated to a pET24a(+) vector (Novagen) treated
with the same enzymes and dephosphorylated. The resulting plasmid
(pET/TXNPx) was used to transform E. coli BL21(DE3).
Transformed cells were selected by kanamycin resistance, the plasmids
purified and sequenced.
E. coli BL21(DE3) pET/TXNPx was grown to OD600
of 0.9-1.0 at 36 °C and 180 rpm in LB medium with 30 µg of
kanamycin/ml, then expression of the tryparedoxin peroxidase gene was
induced with 1 mM
isopropyl-
-D-thiogalactopyranoside. E. coli
BL21(DE3) containing the pET24a plasmid was grown in the same way.
Samples taken at different times were centrifuged, resuspended in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA buffer,
sonicated, and centrifuged. After 5 h the culture was centrifuged
and either stored at
20 °C or the cells were resuspended in 0.05 culture volume of binding buffer (5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9). The cell
suspension was sonicated on ice and centrifuged for 40 min at 4 °C,
35,000 × g. The supernatant was applied to a
His·Bind resin (Novagen) column charged with Ni2+ and
equilibrated with binding buffer, at a flow rate of about 10 column
volumes/h. The column was washed with 10 volumes of binding buffer, 6 volumes of 500 mM NaCl, 20 mM Tris-HCl, pH 7.9, buffer containing 60 mM imidazole and 6 volumes of the same
buffer with 100 mM imidazole. Tryparedoxin peroxidase
eluted in the buffer containing 500 mM imidazole. Active
fractions were pooled and immediately dialyzed against 50 mM Tris-HCl, pH 7.6, buffer containing 100 mM
NaCl and 1 mM EDTA.
Characterization of Recombinant Tryparedoxin
Peroxidase--
SDS-PAGE was done under reducing conditions in 8-25%
gradient gels on a Pharmacia Phast System and the gels were stained for protein with silver according to the manufacturers' recommendations. Western blots were performed with the same system on polyvinylidene difluoride membranes. Whole rabbit serum (1:250 dilution) containing antibodies raised against pure C. fasciculata tryparedoxin
peroxidase was used as primary antibody and anti-rabbit goat antibodies
(Sigma) as secondary antibody. Enzyme activity and kinetic studies were performed as described in Nogoceke et al. (1); protein
concentration was determined using Coomassie Brilliant Blue G reagent
(Bio-Rad) with bovine serum albumin as standard.
Analysis of Molecular Evolution--
The EMBL and SWISSPROT data
bases were screened for tryparedoxin peroxidase homologous nucleotide
or peptide sequences using BLAST and FASTA. Multiple alignments were
performed by the program PILEUP (GCG Wisconsin Program Package, Version
9.0-OpenVMS) using default parameters. The polypeptide sequences of
data base entries comprising complete coding regions were compared by
the program DARWIN (16).
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RESULTS |
Isolation and Sequencing of the Tryparedoxin Peroxidase Gene from
C. fasciculata--
Sequenced peptide fragments obtained from purified
tryparedoxin peroxidase of C. fasciculata could be aligned
along the established deduced amino acid sequence of the thiol-specific
antioxidant protein of yeast (1). This enabled appropriate degenerate
PCR primers to be designed for the generation of a PCR product from the
C. fasciculata genomic DNA. This PCR product, which was 0.4 kb long (see Fig. 3), was subsequently used to screen a genomic library
for inserts containing the full-length DNA encoding the tryparedoxin
peroxidase. A clone containing a 15-kb insert with the presumed
tryparedoxin peroxidase gene was isolated and sequenced. This, however,
led to the detection of equal quantitites of different nucleotides at
several positions toward the 3' end of the gene, implying the presence
of similar but not identical genes in the insert. This observation was
not unexpected since the genome of the Trypanosomatidae is
known to contain repetitive structural genes separated by intergenic
sequences (17-19). The
-clone was consequently digested with the
restriction enzyme SacI and a Southern blot was performed.
Three fragments belonging to the insert (1.1, 1.5, and 11 kb) gave
positive hybridization signals with the labeled PCR product. Each of
the three fragments was subcloned into pBluescript II KS(+/
)
phagemids and sequenced. The general sequencing strategy is shown in
Fig. 2 using the 1.5-kb fragment as an
example.

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Fig. 2.
Restriction map and sequencing strategy for
the tryparedoxin peroxidase gene. The region shown is that of the
1.5-kb fragment. The open reading frame for the tryparedoxin peroxidase gene (TXNPx) is indicated as an open box. The
arrows show the directions and approximate positions of the
primers used for sequencing. The two terminal arrows
indicate primers hybridized to the cloning vector.
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The 11-kb fragment contained the information coding for the previously
sequenced peptides of tryparedoxin peroxidase (Fig. 3). Nevertheless, as with the 15-kb
insert, the bases at positions 542, 548, 551, 556, 557, 560, 563, 564, and 565 remained ambiguous suggesting the presence of more than one
gene in this fragment. The nucleotide sequence shown in Fig. 3 was
confirmed by resequencing the 1.5-kb fragment which contained an open
reading frame largely identical to the one of the 11-kb fragment except
for the presence of an additional cytosine at position 30. As a
consequence of the resulting frameshift, the deduced amino acid
sequence no longer complied with the established peptide sequences.
Hence, the 1.5-kb fragment contained a pseudogene. The 1.1-kb fragment
also contained an open reading frame but encoded only part of the
tryparedoxin peroxidase since a SacI restriction site was
present at position 472-477. This reading frame was therefore not
sequenced to completion. All the amino acid sequences determined for
the peptide fragments of the C. fasciculata tryparedoxin
peroxidase matched those predicted from the DNA shown in Fig. 3, except
for positions 406-408 where an asparagine is encoded but a threonine
was determined by amino acid sequencing. This asparagine was found in
all of the sequenced DNA fragments and in the PCR product.

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Fig. 3.
Nucleotide and deduced amino acid sequences
of the tryparedoxin peroxidase gene from C. fasciculata. The arrows delimit the PCR product
used to screen the genomic library. The start and stop codons are in
bold, as is the asparagine residue which was replaced by a
threonine residue in direct peptide sequencing. Sequences confirmed by
protein sequence analysis are underlined. The position of
the SacI site in the 1.1-kb fragment is heavily underlined. The differences in the coding region between the 1.5- and 11-kb fragments, and in the 5'-flanking region between the 1.5- and
1.1-kb fragments are shown in brackets. The AG consensus splice leader sites and the polypyrimidine-rich tract are double underlined.
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When comparing the 5'-flanking region of the 1.5-kb fragment with one
of the 1.1-kb fragments, only minor differences were found (Fig. 3). In
both fragments two possible consensus dinucleotides AG, which represent
potential splice leader acceptor sites, are found in positions
52 and
125. However, only the one in position
125 is preceded by a
pyrimidine-rich tract which is necessary for trans-splicing (20).
Expression of the Tryparedoxin Peroxidase Gene in E. coli
BL21(DE3)--
The identification of multiple open reading frames
encoding tryparedoxin peroxidase required confirmation of functionality of the sequence shown in Fig. 3. For this purpose the 1.5-kb fragment exhibiting least sequence ambiguities appeared most appropriate except
for the inserted frameshift mutation. Therefore, the frameshifting extra cytosine base at position 30 was deleted, the region coding for
the C-terminal amino acids was modified as described under "Experimental Procedures" to facilitate purification of the
expression product, and the resulting coding region was incorporated
into the expression vector pET 24a to give the pET/TXNPx plasmid (Fig. 4). After induction of the transformed
bacteria, tryparedoxin peroxidase activity was detected in supernatants
of sonicated cells. The activity increased to a maximum 5 h after
induction while no activity was found in the control (Fig.
5). Induction resulted in the
accumulation of a new protein with an apparent molecular mass of 21,000 in SDS gels which was recognized by the anti-tryparedoxin peroxidase
antibodies (Fig. 6B) and
proved to be an active tryparedoxin peroxidase.

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Fig. 4.
Structure of the plasmid vector used for the
expression of tryparedoxin peroxidase in E. coli. The
restriction sites (XhoI and NdeI) where the
tryparedoxin peroxidase gene (TXNPx) was inserted are shown.
His, histidine tag coding sequence; Kan r,
kanamycin resistance coding sequence; lacI, LacI coding
sequence; ori, pBR322 origin; T7 prom, T7
promoter.
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Fig. 5.
Tryparedoxin peroxidase activity in the
supernatants of E. coli pET24 and E.coli
pET/TXNPx. LB medium containing 30 µg of kanamycin/ml was
inoculated with a single colony and the E. coli cells were
grown at 36 °C, 180 rpm. When OD600 was 0.3 (time 0),
10-ml aliquots were withdrawn at hourly intervals, resuspended in 2 ml
of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA buffer, sonicated, and centrifuged. Tryparedoxin peroxidase activity was measured in the supernatant as stated under "Experimental
Procedures." The arrow indicates the time of induction by
isopropyl- -D-thiogalactopyranoside addition. ,
E. coli pET24; , E. coli pET/TXNPx.
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Fig. 6.
Analysis of tryparedoxin peroxidase expressed
in E. coli by SDS-PAGE and Western blotting. A,
SDS-PAGE was done under reducing conditions in 8-25% gradient gels on
a Pharmacia Phast System and the proteins were visualized by silver
staining according to the manufacturers' recommendations. Lane
1, supernatant of E. coli BL21(DE3) pET/TpodH6 cells
before induction; lane 2, supernatant of E. coli
BL21(DE3) pET/TpodH6 cells 5 h after induction; lane 3,
purified recombinant tryparedoxin peroxidase; lane 4, authentic tryparedoxin peroxidase from C. fasciculata;
lane 5, molecular weight standards. B, Western
blotting was performed by electrotransferring proteins from SDS gels
onto polyvinylidene difluoride membranes. Whole rabbit serum (1:250
dilution) containing antibodies raised against pure C. fasciculata tryparedoxin peroxidase was used as primary antibody
and anti-rabbit goat antibodies (Sigma) as secondary antibody for the
immunodetection of recombinant tryparedoxin peroxidase. Lane
1, supernatant of E. coli BL21(DE3) pET/TpodH6 cells
before induction; lane 2, supernatant of E. coli
BL21(DE3) pET/TpodH6 cells 5 h after induction; lane 3,
purified recombinant tryparedoxin peroxidase.
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Purification and Characterization of the Recombinant Tryparedoxin
Peroxidase--
The addition of 6 histidine residues to the C-terminal
end of the expressed protein enabled simple purification of the
recombinant tryparedoxin peroxidase on a Ni2+-binding
resin. Tryparedoxin peroxidase bound to the resin, whereas most of the
impurities either did not bind or were washed from the column with the
binding buffer containing 100 mM imidazole. Tryparedoxin
peroxidase eluted at 500 mM imidazole and was shown to be
homogeneous by SDS-PAGE and subsequent silver staining (Fig. 6A). N-terminal sequencing of this protein showed the
initial methionine to be missing and allowed us to confirm the first 30 amino acids. The expressed tryparedoxin peroxidase had nearly the same
molecular mass of about 21,000 as the authentic tryparedoxin peroxidase
in SDS-PAGE (Fig. 6A). Matrix-assisted laser
desorption/ionization-mass spectrometry analysis demonstrated the
molecular mass of the recombinant enzyme to be 21,884 ± 22. The
difference in the molecular mass of 1,004 to the authentic tryparedoxin
peroxidase (1) corresponded to the additional amino acids (leucine,
glutamate, and 6 histidines) added at the C-terminal end of the
recombinant enzyme. Whereas the authentic tryparedoxin peroxidase
contained several isoforms ranging from pI 4.9 to 5.8, the recombinant
protein showed two bands of pI 6.2 and 6.3. The higher alkalinity
results from the additional histidines residues.
The pure recombinant enzyme had a specific activity of 2.51 units/mg
compared with 5.83 units/mg for the authentic enzyme. This difference
may be due to the additional residues at the C-terminal end of the
recombinant tryparedoxin peroxidase. The kinetic analysis of the
recombinant protein revealed a kinetic pattern identical to that of the
authentic tryparedoxin peroxidase, i.e. a ping-pong mechanism with infinite maximum velocities and Michaelis constants. Initial velocities when fitted to the general Dalziel equation for
bisubstrate reactions, where ROOH = hydroperoxide and
TXN = tryparedoxin,
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(Eq. 1)
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revealed that the coefficients
0 and
1, 2 were zero. The kinetic parameters obtained with
the pure recombinant tryparedoxin peroxidase and the authentic
tryparedoxin peroxidase isolated from C. fasciculata are
compared in Table I.
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Table I
Dalziel coefficients ( 1 and 2) and apparent
second-order rate constants (k'1 and k'2) for the
reactions of recombinant and authentic tryparedoxin peroxidases with
t-butyl hydroperoxide ( 1 and k'1) and tryparedoxin
( 2 and k'2)
Data are the means of six independent measurements. All values are
calculated per subunit concentration. The parameters for the authentic
enzyme are taken from Nogoceke et al. (1).
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Comparison with Homologous Sequences--
Based on sequence
analysis, tryparedoxin peroxidase belongs to the family of
peroxiredoxins (1). More than 50 homologous sequences were detected in
the SwissProt and EMBL data banks, which comprised various bacterial
alkyl hydroperoxide reductases (5), the thioredoxin peroxidases from
yeast (21) and mammals (4) previously called TSA proteins (22-24),
NKEF-
and -
(25), a closely related gene of Trypanosoma
brucei rhodesiense (26), and a large number of genes with still
undefined functions.
Out of these protein and DNA sequences 47 were selected to construct an
unrooted phylogenetic tree, whereby the selection was based on (i)
completeness of the published sequence, (ii) similarity in size, and
(iii) likelihood of homology with a maximum FASTA E-score of 4.5. In
the thereby obtained phylogenetic tree (Fig.
7), tryparedoxin peroxidase
(Q) together with the homologous T. brucei
sequence (R) presents a distinct molecular clade branching off between the sequences of the metazoa (A-P), including
one of the known helminth sequences (T) on the one side with
the rest, comprising the sequences of entamoeba (S), yeast
(
,
), plants (
-µ), bacteria (a-o), and,
surprisingly, another helminth (U), on the other side.

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Fig. 7.
Phylogenetic tree of the peroxiredoxin family
of proteins constructed by the program DARWIN. = higher
animals: A, osteoblast-specific factor 3 (OSF3) Mus
musculus (accession number D21252); B, 23-kDa
stress-induced peritoneal macrophage protein (MSP23) M. musculus (AC number D16142); C, heme-binding protein (HBP) Rattus norvegicus (AC number D30035);
D, proliferation-associated gene (PAG) H. sapiens (AC number X67951); E, natural killer enhancing
factor A (NKEF- ) H. sapiens (AC number L19184); F, animal blastomere protein (ABP-25) Cynops
pyrrhogaster (AC number D37808); G, natural killer
enhancing factor-like protein (RBT-NKEF) Oncorhynchus mykiss
(AC number U27125); H, antioxidant enzyme (AOE37-2) H. sapiens (AC number U25182); I, TSA R. norvegicus (AC number U06099); J, TSA (thioredoxin
peroxidase, TPX) M. musculus (AC number X82067,
U51679); K, thioredoxin-dependent peroxide
reductase (TPX2) M. musculus (AC number U20611);
L, natural killer enhancing factor B (NKEF- ) H. sapiens (AC number L19185); M, TSA H. sapiens (AC number Z22548); N, erythroleukemia antisense RNA (MER5 = AOP1) M. musculus (AC number
M28723); O, antioxidant protein (AOP) and
substrate protein for mitochondrial ATP-dependent protease
Bos taurus (AC number D82025); P, AOP H. sapiens (AC number D49396). ,
nematodes: T, TSA B. malayi (AC number U34251);
U, TSA Onchocerca volvulus (AC number P5250). , protozoa: Q, tryparedoxin peroxidase (TXNPx)
C. fasciculata (AC number AF020947); R, alkyl
hydroperoxide reductase (AHR) T. brucei
rhodesiense (AC number U26666); S, 29-kDa cysteine-rich surface antigen (CR29) Entamoeba histolytica (AC number
P19476). , yeast: , TSA S. cerevisiae (AC number
L1640); , 29.5-kDa protein in UBP13-KIP1 intergenic region (YBG4)
S. cerevisiae (AC number P34227). , plants: ,
peroxide reductase (BAS1) Spinacea oleracea (AC number
X94219); , BAS1 Arabidopsis thaliana (AC number Y10478);
, TSA Triticum aestivum (AC number AB000405); , TPX-
and AHR-like protein (BAS1) Hordeum vulgare (AC number Z34917); , alkyl hydroperoxidase C (AHPC)/TSA-like 22.3-kDa protein
(YC42) Porphyra purpurea (AC number P51272); , 23.5-kDa protein (YCF42) Odonthella sinensis (AC number P49537); , abscisic acid (ABA) responsive 24-kDa polypeptide (RAB24)
Oryza sativa (AC number D63917); , ABA responsive gene
(ABAR) Bromus secalinas (AC number X63202); µ, rehydrin
(REHY) Tortula ruralis (AC number U40818). , bacteria:
a, 20-kDa protein in rubredoxin (RUBR) Clostridium
pasteurianum (AC number P23161); b, species-specific protein antigen (26 kDa) Heliobacter pylori (AC number
M55507); c, AHPC C22 protein B.
subtilis (AC number P80239); d, AHPC C22 protein
S. typhimurium (AC number P19479); e, AHPC C22 protein E. coli (AC number P26427); f,
bacterioferritin comigratory protein (BCP) E. coli (AC
number P23480); g, BCP Hemophilus influenzae (AC
number P44411); h, 18-kDa protein in Streptococcus sanguis adhesinB gene S. sanguis (AC number 31308);
i, 18-kDa protein in S. gordonii coaggregation
mediating adhesin 3' region orf4 S. gordonii (AC number
P42366); j, 18-kDa protein in fibrial protein-adhesin 3'
region orf3 S. parasanguis (AC number P31307); k,
AHR/TSA homolog gene Sulfolobus sp. (AC number U36479); l, AHPC (= Mycobacterium avium antigen, AVI3)
M. avium (AC number U18263); m, AHPC M. tuberculosis (AC number U18264); n, AHPC M. smegmatis (AC number U43719); o, iron-repressible
polypeptide (DIRA)/AHPC C. diphtheriae (AC number
U18620). The addition of numbers shown in each branch of the tree
allows calculation of the estimated pam distance between sequence
pairs.
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Sequence alignments were further used to generate ideas about the
potential functional relevance of particular amino acid residues. For
this purpose we aligned a total of nine sequences for which a
peroxidase function had been either clearly established or suggested
from biological experiments (Fig. 8).
These were the thioredoxin peroxidases of Saccharomyces
cerevisiae (21) and Homo sapiens (4), the tryparedoxin
peroxidase described here, and the alkyl hydroperoxide reductases from
Salmonella typhimurium (5), E. coli
(27), Corynebacterium diphtheriae (28), Bacillus subtilis (29), and Mycobacterium tuberculosis (30).
Although the similarities between these sequences of poorly related
species ranged from 29 to 77%, a total of 21 residues were strictly
conserved in homologous positions. The overall consensus sequence is:
xnAx5-6Fx9Gx3Vx2Fx1Px2Fx1FVCPTEx21Sx1Dx7Wx16-19Dx15-16Gx3Rx2Fx2Dx27AxQx4-11Cx1-3Wxn.

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Fig. 8.
GCG alignment of peroxiredoxins with known
peroxidase activity. Gap creation penalty = 12; gap extension
penalty = 4. Strictly conserved amino acids and the highly
conserved ones forming the degenerate second VCP motif are marked in
bold. AHPC, alkyl hydroperoxidase C,
TXNPx, tryparedoxin peroxidase; Bsub, B. subtilis;
Cfas, C. fasciculata; Cdip, C. diphtheriae; Ecol, E. coli, Hsap, H. sapiens; Mtub, M. tuberculosis; Scer, S. cerevisiae; Styp, S. typhimurium.
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The first cysteine residue is integrated into an absolutely conserved
VCP motif which is widespread in the whole peroxiredoxin family. The
C-proximal cysteine also forms a VCP motif in seven of the aligned
peroxidases, whereas in M. tuberculosis the motif is altered
to LCA. The C-proximal VCP and LCA motifs, respectively, are separated
by two residues from the last conserved tryptophan. In the thioredoxin
peroxidase of yeast the corresponding region reads VLPCNW.
 |
DISCUSSION |
We have cloned a genomic DNA fragment from C. fasciculata that encodes multiple copies of the entire sequence of
tryparedoxin peroxidase, which is an essential constituent of the
trypanosomatidal peroxide metabolism (1). The reading frame is very
similar to a DNA sequence of T. brucei rhodesiense encoding
a protein of unknown function (26). The high degree of similarity
suggests that this trypanosomal protein is also a tryparedoxin
peroxidase. However, the multiplication of genes in the
trypanosomatids, which may lead to pseudogenes, precludes a definite
functional interpretation of DNA sequences without experimental
evidence. In the case of the C. fasciculata DNA sequence
shown in Fig. 3, its functional relevance was unambiguously established
by heterologous expression and comparison of the gene product with the
authentic tryparedoxin peroxidase.
Tryparedoxin peroxidase unequivocally belongs to the family of
peroxiredoxins. This protein family is obviously widespread in nature.
The phylogenetic tree showing the molecular evolution of the
peroxiredoxins practically covers all phyla from bacteria to
vertebrates but it does not simply reflect the phylogenetic divergence
of the species. The sequence of the parasitic plathelmint Brugia
malayi (T), for example, debranches close to protozoal and
vertebrate sub-trees, whereas another plathelmint protein (U) belongs
to a plant sub-tree. There are two distinct molecular clades found in
chlorophyta plus a separate type in the diatomean alga
Odonthella, each diverging at distant points from bacterial branches. Similarly, one of the two yeast peroxiredoxins debranches from a plant branch, which in turn diverges from bacterial ancestors. This puzzling situation suggests multiple gene acquisition by means of
endosymbiosis. Such gene transfer has also been implicated for various
Euglenazoa, including the Trypanosomatidae (31). Even the cytosolic GapC of T. brucei and
Leishmania mexicana, for example, is believed to be acquired
from endosymbiotic
-purple bacteria and to be secondarily integrated
into the nuclear genome of the trypanosomatids. The debranching point
of the two trypanosomatid peroxiredoxins, however, does not lend any
support to speculations about secondary gene acquisition. Debranching
between the metazoa and yeast, bacteria, and plants, the molecular
evolution of the trypanosomal peroxiredoxins appears congruent with
taxonomical development.
Molecular evolution has given ample room for functional diversification
within the peroxiredoxin family and it can by no means be uncritically
presumed that all peroxiredoxins are peroxidases or "antioxidant
proteins." Conceivably, many of the peroxiredoxins will turn out to
reduce hydroperoxides, but the reducing substrates differ substantially
in the examples investigated to date. TSA of yeast, like human NKEF-
and -
, utilizes thioredoxin as a reductant; the crithidial homolog
is reduced by a remote relative of thioredoxin, tryparedoxin, which is
50% larger than the typical thioredoxins and is characterized by a
WCPPC motif in its active site; the alkyl hydroperoxide reductases of
bacteria are directly reduced by flavoproteins also containing vicinal
thiols (AhpF); and from their history of discovery we know that the
activity of TSAs depends on dithiols such as dithiothreitol. The common denominator of the peroxidase activity of peroxiredoxin thus appears to
be the regeneration by dithiols of a residue reacting with hydroperoxides. In the absence of any reasonable alternative such a
redox active residue can only be a cysteine. In the case of thioredoxin
peroxidase of yeast, the active site was identified by site-directed
mutagenesis as the N-proximal VCP motif (32). Interestingly,
replacement of the conserved C-terminal cysteine residue by a serine
resulted in an active antioxidant protein when tested with the
non-physiological substrate dithiothreitol, but the activity with
thioredoxin was lost (21, 32). With tryparedoxin peroxidase, the
participation of two cysteine residues in the catalysis was
demonstrated by the substrate-dependent inactivation with
N-ethylmaleimide and confirmed by matrix-assisted laser
desorption/ionization-time of flight-mass spectrometry (1). The
consensus sequence of the peroxiredoxins with experimentally
demonstrated peroxidase activity reveals two conserved cysteines as the
only potentially redox-active functional groups. Only the first
cysteine appears to be obligatorily integrated into a VCP motif within
a highly conserved region. Although, the second cysteine also usually
forms a VCP motif, it appears to tolerate substantial changes in its intimate sequence context. However, based on the yeast TSA example, it
is tempting to speculate that the latter is crucial for the reaction
with the specific reductant, whereas the former is indispensible for
the reaction with the oxidant.
The kinetics of tryparedoxin peroxidase have been elucidated in detail
(1). The apparent net forward rate constants for the reaction of the
reduced enzyme (calculated per subunit) with a variety of
hydroperoxides were found to be around 105
M
1 s
1. To achieve such rate
constants the active site cysteine residue must be highly activated.
These rate constants are virtually identical to those of sulfur
homologs of the selenocysteine containing glutathione peroxidases (33,
34), where the (seleno)cysteine residue, together with a tryptophan and
a glutamine residue, forms a unique catalytic triad and thereby is
forced into full dissociation and is correspondingly reactive (34).
Interestingly, two tryptophans and one glutamine are also strictly
conserved in the peroxiredoxin-type peroxidases and it may therefore be
speculated that their cysteines might be activated in an analogous way
to the structurally unrelated glutathione peroxidases. In fact, with
the exception of a single arginine, none of the other residues of the
consensus sequence would be able to force a cysteine into dissociation.
The three aspartate residues, if situated near the active site
cysteine, would be contraproductive, the lipophilic residues would be
largely indifferent, and the conserved prolines and glycines are most probably involved in the preservation of the three-dimensional structure. Unfortunately, not a single three-dimensional structure of a
peroxiredoxin is presently available to confirm the hypothesis of the
convergent evolution of an analogous active site by unrelated proteins.
We thank Dr. Michael Kie
and Rita Getzlaff
for amino acid sequencing.