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Volume 272, Number 49, Issue of December 5, 1997
pp. 30841-30847
(Received for publication, July 23, 1997, and in revised form, September 26, 1997)
From the Department of Biosciences at Novum, Karolinska Institute,
S-141 57 Huddinge, Sweden
Thioredoxin (Trx) is a small ubiquitous protein that
displays different functions mainly via redox-mediated processes. We here report the cloning of a gene (trxC) coding for a novel
thioredoxin in Escherichia coli as well as the expression
and characterization of its product. The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa. Trx2
contains two distinct domains: an N-terminal domain of 32 amino acids
including two CXXC motifs and a C-terminal domain, with the
conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to
the prokaryotic thioredoxins. Trx2 together with thioredoxin reductase
and NADPH is an efficient electron donor for the essential enzyme
ribonucleotide reductase and is also able to reduce the interchain
disulfide bridges of insulin. The apparent Km value
of Trx2 for thioredoxin reductase is similar to that of the previously
characterized E. coli thioredoxin (Trx1). The enzymatic
activity of Trx2 as a protein-disulfide reductase is increased by
preincubation with dithiothreitol, suggesting that oxidation of
cysteine residues other than the ones in the active site might regulate
its activity. A truncated form of the protein, lacking the N-terminal
domain, is insensitive to the presence of dithiothreitol, further
confirming the involvement of the additional cysteine residues in
modulating Trx2 activity. In addition, the presence of the N-terminal
domain appears to confer heat sensitivity to Trx2, unlike Trx1.
Finally, Trx2 is present normally in growing E. coli cells
as shown by Western blot analysis.
Thioredoxin (Trx)1 is a
small protein (Mr 12,000) with a conserved
active site sequence Trp-Cys-Gly-Pro-Cys that catalyzes many redox
reactions through the reversible oxidation of its active site dithiol
to a disulfide. Oxidized thioredoxin, Trx-S2, can be
reduced by NADPH and the flavoenzyme thioredoxin reductase, the
so-called thioredoxin system (Reaction 1) (1). Reduced thioredoxin,
Trx-(SH)2, contains two thiol groups and can efficiently catalyze the reduction of many exposed disulfides, thus being a
general protein-disulfide reductase, Reaction 2.
E. coli Trx is a well studied enzyme, and its
three-dimensional structure has been determined by NMR for both
oxidized and reduced forms, as well as by x-ray crystallography (17,
18). E. coli thioredoxin contains 109 amino acids and has a
central core of five strands of twisted Thioredoxin-negative mutants (trxA We report here the cloning of a DNA sequence coding for a novel E. coli thioredoxin (Trx2) based upon biological activity data and protein homology. We also present evidence that the protein is normally expressed in E. coli cells and that the N-terminal sequence of the protein contains a novel domain with four cysteine residues that partly regulates its enzymatic activity as protein-disulfide reductase. Strains and Media E. coli K-12 was a stock from our laboratory. K38 (wild type) and A179 (trxA::kan) (27) strains were a kind gift from Prof. Arne Holmgren (Karolinska Institutet, Stockholm). Cells were grown in LB medium supplemented (when necessary) with 50 µg/ml ampicillin or kanamycin. Cloning of the E. coli Thioredoxin 2 A thioredoxin-like sequence (Trx2) containing an open reading
frame coding for a protein of 139 amino acids (GenBankTM
accession number 1788936),2 was
used to design the specific mutagenic primers
EcTrx2-NdeI, 5 Protein Expression and Purification The insert of Trx2 cloned into the pGEM-T vector was digested
with NdeI and BamHI and cloned into the
NdeI/BamHI sites of the pET-15b expression vector
(AMS Biotechnology). The recombinant plasmid was designated pET-Trx2.
E. coli BL21 (DE3) was transformed with the pET-Trx2
construct, and a single positive colony was inoculated in 1 liter of LB
medium plus ampicillin and grown at 37 °C until
A600 = 0.5. Then fusion protein was induced by
the addition of 0.5 mM
isopropyl-1-thio- The truncated form of Trx2 ( Antibodies and Immunoblotting Analysis For antibody production, we used a modification of the method described by Song et al. (28). Female chickens were injected (once every 15 days during 6 weeks) subcutaneously at multiple sites with 100 µg (in 0.25 ml) of His-Trx2 in complete adjuvant. After the second injection, eggs were collected daily, and when a suitable number of eggs were obtained antibodies were purified as follows. Egg yolks were separated from whites by dropping the egg contents on a funnel placed in a graduated cylinder. An equal volume of buffer S (10 mM phosphate, pH 7.5, 0.1 M NaCl, containing 0.01% sodium azide) was added to the yolks and stirred. Next, 10.5% PEG 8000 (Sigma) in buffer S was added to a final concentration of 3.5%. The mixture was stirred for 30 min at room temperature and centrifuged at 12,000 × g for 20 min. The supernatant was filtered through two layers of 3MM Whatman chromatography paper, and 42% PEG 8000 in buffer S was added to a final concentration of 12%. The mixture was stirred thoroughly and centrifuged at 12,000 × g for 20 min. The supernatant was discharged, and the pellet was redissolved in 12% PEG in buffer S to the original yolk volume. After centrifugation, the pellet was dissolved in 30 ml of buffer S and dialyzed overnight against buffer S without NaCl. Affinity-purified antibodies were prepared using a cyanogen bromide-activated Sepharose 4B column where 1 mg of Trx2, in which the His tag had been removed by thrombin, had been coupled following the procedure recommended by the manufacturer (Pharmacia Biotech Inc.). The specificity of the antibodies was tested by Western blotting using recombinant Trx2 and total cell extracts. For immunoblotting, samples were subjected to 15% SDS-polyacrylamide gel electrophoresis, and the separated proteins were electrophoretically transferred to nitrocellulose membranes (Hybond-C Super, Amersham Corp.). The membranes were blocked with phosphate-buffered saline containing 8% dry fat-free milk powder, 2% bovine serum albumin (BSA), 150 mM NaCl, and 0.1% Tween 20 and further incubated with affinity-purified anti-Trx2 antibodies. Immunodetection was performed with horseradish peroxidase-conjugated rabbit anti-chicken IgG (Sigma) diluted 1:5000, following the ECL protocol (Amersham). Preparation of Cellular Fractions from E. coli Lysozyme TreatmentCells were harvested at A600 = 0.5 and washed three times in 50 mM Tris, pH 8.0. The pellet was resuspended in the same buffer plus 2 mM MgCl2 and 2 mg/ml lysozyme at an A600 = 40, incubated for 30 min on ice, and centrifuged at 14,000 rpm at 4 °C for 1 min. The supernatant after centrifugation was collected as the periplasmic lysate fraction. The pellet was resuspended in the same volume of 50 mM Tris, pH 8.0, 2 mM MgCl2 and kept as the cytosolic fraction. Freeze-Thaw TreatmentCells were harvested and washed as in the previous treatment. The pellet was resuspended in 50 mM Tris, pH 8.0, 2 mM MgCl2 at the same A600 and quickly frozen in a dry ice/ethanol bath. The frozen suspension was slowly thawed in an ice bucket, and this cycle was repeated three times. The suspension was centrifuged, and periplasmic and cytosolic fractions were collected as described above. For crude bacteria extracts, exponentially growing cells were harvested by centrifugation, and the pellet was resuspended in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA. Cells were sonicated, and the supernatant was cleared by centrifugation for 15,000 × g at 4 °C for 30 min. Heated extracts were prepared by heating crude extracts for 5 min, placing them on ice and spinning as described above. Enzyme Assays of E. coli Thioredoxin 2 The activity of E. coli Trx2 was determined by the
DTNB and insulin assays. The DTNB assay was performed essentially as
described elsewhere (29). Briefly, the reaction mix contained 200 mM phosphate buffer, pH 7.0, 2 mM EDTA, 0.1 mg/ml BSA, 1 mM DTNB, and 0.5 mM NADPH. The
reactions containing 0.5-8 µM Trx1, Trx2, or Ribonucleotide Reductase Assay The ability of E. coli Trx1 and Trx2 to serve as hydrogen donor for NrdAB and EF reductases was determined using the standard ribonucleotide reductase assay (30). Stoichiometric amounts (1 µM final concentration) of R1A/R2B and R1E (1.8 µg)/R2F (0.6 µg) were incubated for 20 min at 37 °C with Trx1 (IMCO) or Trx2 in a final volume of 50 µl containing 0.5 mM [3H]CDP (19,100 cpm/nmol), 10 mM MgCl2, 50 mM Tris-HCl, pH 8.0, 1 mM NADPH, 0.1 µM E. coli thioredoxin reductase, and 0.3 mM dATP (for NrdEF) or 1.5 mM ATP (for NrdAB). The reaction was stopped with 0.5 ml of HClO4, and the [3H]dCDP formed was determined by liquid scintillation after ion exchange chromatography on Dowex 50 columns. One enzyme unit is the activity that produces 1 nmol of dCDP during 1 min under these conditions. Cloning of E. coli Trx2 The identification of a novel thioredoxin sequence in the E. coli genome appeared serendipitously when using the recently described mammalian Trx2 (31) cDNA as probe in a Northern blot analysis with E. coli genomic DNA as a negative control. The strong signal obtained (data not shown) prompted us to search for the tentative E. coli homologue of the mammalian Trx2. We identified a DNA sequence in the E. coli genome (not completed at the time of this search) that displayed high homology with mammalian Trx2, but when translated to protein in the three possible reading frames only short peptide sequences were obtained. These short peptide sequences could be due to a possible sequencing mistake (an additional deoxycytidine) that, when removed, resulted in an open reading frame of 417 base pairs encoding a protein of 139 amino acids with the typical thioredoxin active site WCGPC. To further confirm the existence of this sequence coding for a new thioredoxin in the E. coli genome, we used two primers, EcTrx2-NdeI and EcTrx2-BamHI (see "Materials and Methods") to amplify a fragment of 420 base pairs from genomic E. coli K-12 DNA. This PCR fragment was further cloned into pGEM-T Easy vector, and sequencing confirmed the absence of the additional deoxycytidine. Sequence Analysis and ComparisonThe sequence of the Trx2 is
positioned at min 58.5 in the E. coli chromosome map
downstream of the uracyl DNA glycosylase gene. The nucleotide sequence
of the E. coli Trx2 gene and flanking regions is given in
Fig. 1 together with the deduced amino acid sequence. The open reading frame extends from the methionine codon, ATG, at coordinate +1 to the termination codon, TAA, at coordinate 417. This region codes for a protein of 139 amino acids with a estimated
molecular mass of 15.5 kDa, containing the classical active site of
thioredoxins WCGPC. The methionine +1 codon is preceded by a potential
ribosome binding site at position Fig. 1. Nucleotide and deduced amino acid sequence of the trxC gene of E. coli and flanking regions. Nucleotides are numbered (left) beginning with the first base of the ATG initiator codon. Amino acid residues are numbered (left, in parentheses) beginning with the initiating methionine. The ribosome binding site, the upstream TAA stop codon, and tentative 10 and 35 regions are overlined. The active site WCGPC and the four cysteine
residues are boxed. The putative Rho-independent terminator
downstream from the stop codon is centered at position 437 and
indicated by inverted arrows. The ATG codon for the
hypothetical 27-kDa protein (Hyp) downstream trxC
gene is also boxed.
[View Larger Version of this Image (36K GIF file)]
The main difference in Trx2 protein sequence with respect to the well
known Trx1 is the presence of an extra stretch of 32 amino acids at the
N terminus. The C-terminal half of the protein contains the active site
found in all thioredoxins (Fig. 2). Recently, Lim et al. (34) have described a third thioredoxin in
Corynebacterium nephridii, which displays a similar
structure to E. coli Trx2 with an extra N-terminal domain of
32 amino acids and a C-terminal domain homologous to the rest of the
prokaryotic thioredoxins (Fig. 2). E. coli Trx2 displays a
29% identity with E. coli Trx1 and 37% with C. nephridii Trx3 and is less similar to the mammalian Trx1 and Trx2.
A phylogenetic analysis of several thioredoxins places E. coli Trx2 and C. nephridii Trx3 in the same branch of the tree between E. coli Trx1 and mammalian Trx2 (data not
shown).
Fig. 2. Alignment of predicted amino acid sequence of E. coli Trx2 with E. coli Trx1 (49) and C. nephridii Trx3 (34). Sequences were aligned at the active site disulfide. Identical residues are indicated in black boxes. Cysteine residues out of the active site are in empty boxes. The sequence of E. coli Trx2 was used as reference for the identity/similarity values. [View Larger Version of this Image (24K GIF file)] Enzymatic Activities of E. coli Thioredoxin 2 To establish
that the putative Trx2 does in fact encode a protein with thioredoxin
activity, we cloned the Trx2 gene into the pET-15b expression vector
under the control of a T7 promoter. The resulting plasmid pET-Trx2 was
transformed in E. coli BL21 (DE3), and the expression of
His-Trx2 was induced by the addition of
isopropyl-1-thio- Fig. 3. Activity assay of Trx1 and Trx2. E. coli Trx1 and Trx2 were assayed for their ability to reduce disulfide bonds of insulin. , Trx1; , Trx2. Values are the
average of two measurements. Similar results were obtained in three
independent experiments. The inset shows recombinant Trx2
eluted from Talon column with 50 mM imidazole.
[View Larger Version of this Image (26K GIF file)]
As shown in Fig. 3, oxidized Trx2 was active as a disulfide reductase; however, it exhibited approximately 5-fold lower activity than Trx1 at concentrations between 0.2 and 0.5 µM and 1.5-2-fold lower activity between 0.5 and 2 µM. At these higher thioredoxin concentrations, the efficiency of thioredoxin reductase is the rate-limiting step, since the reactivity of Trx-(SH2) and insulin is known to be very fast (35, 36). To understand the role of the extra cysteines present at the N terminus
of Trx2, we preincubated Trx2 with DTT. As shown in Fig.
4, the ability of Trx2 to reduce insulin was
increased to levels similar to that of Trx1 (compare with Fig. 3). The
fact that Trx2 activity was enhanced after reduction of the protein suggested that cysteine residues in Trx2 other than those in the active
site could be involved in regulating its enzymatic activity. We used
the truncated form of Trx2 ( Fig. 4. Effect of DTT on Trx2 insulin-reducing activity. DTT preincubation of different amounts of Trx2 and Trx2 was carried out by adding 2 µl of DTT buffer (50 mM HEPES, pH 7.6, 100 µg/ml BSA, and 2 mM
DTT) in a final volume of 20 µl and incubating for 20 min at
37 °C. Then reaction mix and E. coli thioredoxin
reductase were added to a final volume of 60 µl to initiate the
reaction. The reaction was stopped after 20 min by the addition of 6 M guanidine HCl, 1 mM DTNB. , Trx2 plus DTT;
, Trx2-DTT; , Trx2 plus DTT; , Trx2-DTT. Values are the
average of two measurements. Similar results were obtained in three
independent experiments.
[View Larger Version of this Image (18K GIF file)]
DTNB is an artificial disulfide substrate and a fast oxidant of
Trx-(SH)2, which keeps the concentration of
Trx-S2 constant. DTNB is often used in the assay of
thioredoxin reductase, where one molecule of DTNB is split into two
5
E. coli contains genetic information for
three different ribonucleotide reductases. The NrdAB is active during
aerobiosis. NrdDG is active during anaerobiosis and uses formate as an
electron donor. NrdEF is a cryptic enzyme that uses Grx1 but not Trx1
as an electron donor (37-39). The viability of a triple mutant Trx1, Grx1 and Grx3, the three known electron donors for the NrdAB protein when growing in aerobiosis, prompted us to assay Trx2 as a tentative reductant for this enzyme. As shown in Fig.
5, Trx2 was found to be a functional electron
donor for the NrdAB enzyme. However, compared with Trx1, Trx2 was
almost 2.5-fold less efficient as an electron donor than Trx1. Similar
to Trx1, Trx2 was not an electron donor for NrdEF (40).
Fig. 5. Activity of Trx2 as electron donor for ribonucleotide reductases. The assay was performed with [3H]CDP as substrate in the presence of NADPH, thioredoxin reductase, and increasing concentrations of Trx1 and Trx2 as described under "Materials and Methods." , Trx1 activity with
NrdAB; , Trx2 activity with NrdAB; , Trx2 activity with NrdEF.
Values are the average of two measurements in two independent
experiments.
[View Larger Version of this Image (17K GIF file)] Expression of E. coli Thioredoxin 2 To assay for the presence
of Trx2 activity in E. coli, we used protein extracts from
K38 (wild type) and A179 (trxA::kan) strains (27).
To decrease the background in the insulin assay due to interaction of
DTNB with -SH groups, we used heated extracts (85 °C, 5 min).
Thioredoxin activity was detected in E. coli A179 (trxA Affinity-purified polyclonal antibodies were used to further analyze
the presence of Trx2 in E. coli extracts. Fig.
6A shows that the antibodies
reacted with one band at the expected position of Trx2 in a total crude
extract of E. coli (lane 3), indicating that the
protein is normally expressed. These antibodies also reacted with
E. coli Trx1, being detectable in the wild type strain but
not in the mutant (Fig. 6A, compare lane 3 with
lane 4). However, antibodies raised against E. coli Trx1 did not cross-react with recombinant Trx2 (data not
shown). The affinity of the anti-Trx2 antibodies for Trx1 was 10-fold
lower than for Trx2 (Fig. 6B). From the immunoblot and
densitometric analysis, the contents of Trx2 and Trx1 in the total
crude extract of E. coli were estimated to be about 0.33 and
1.7 µg/mg protein, respectively. The calculated value for Trx1 is in
good agreement with the amount of Trx1 (1.91 µg/mg protein) obtained
by enzyme-linked immunosorbent assay (41).
Fig. 6. Immunoblotting analysis. Samples were separated by SDS-polyacrylamide gel electrophoresis (15%), blotted onto a nitrocellulose membrane, and probed with anti-Trx2 affinity-purified antibodies. A, identification of Trx2 in cell extracts of E. coli. Lane 1, E. coli Trx1 (50 ng); lane 2, E. coli Trx2 (5 ng), where the His tag was removed by thrombin; lane 3, K38 (wild type) total crude extract (15 µg); lane 4, A179 (trxA ) total crude extract (15 µg);
lane 5, pellet after freeze-thaw treatment (5 µg);
lane 6, supernatant after freeze-thaw treatment (5 µg);
lane 7, pellet after lysozyme treatment (10 µg);
lane 8, supernatant after lysozyme treatment (7 µg).
B, affinity of anti-Trx2 antibodies for Trx1 (50, 100, and
150 ng) and Trx2 (5, 10, and 15 ng). Gel B was run and
developed in parallel with gel A and was used for the
quantitation of Trx2. Similar results were obtained from three
independent experiments.
[View Larger Version of this Image (76K GIF file)]
Thus, the levels of Trx2 versus Trx1 were different when
calculated from the Western blots (5-fold lower) than from the activity assays (15-fold lower). One possible explanation is the inactivation of
Trx2 during the preparation of the heated bacteria extracts. Trx1 is a
heat-stable protein, and heated extracts at 85 °C have been used
previously to measure thioredoxin activity. We compared the heat
stability of Trx2 versus Trx1 (Fig.
7). As expected, the activity of Trx1 was not
affected after 5 min of treatment at increasing temperatures from 37 to
85 °C. In contrast, the activity of Trx2 was more dependent on
temperature treatment, and after 5 min at 85 °C 40% of the
insulin-reducing activity was lost. When the N-terminal part of Trx2
was deleted, the heat stability of Trx2 was similar to Trx1, indicating
that the N-terminal part is responsible for the partial heat
inactivation. Treatment with DTT after the heat treatment seems to
restore protein activity.
Fig. 7. Rate of insulin reduction as a function of temperature. 0.5 µg of Trx1, Trx2, and Trx2 were incubated
for 5 min at different temperatures and then analyzed with the insulin
assay. Two separate measurements were made for each protein, and the average values are shown. , Trx2; , Trx2 plus DTT; , Trx2; , Trx1.
[View Larger Version of this Image (18K GIF file)] Subcellular Localization of E. coli Thioredoxin 2 The N-terminal part of Trx2 does not match any consensus sequence for protein translocation to the periplasmic space. To determine whether Trx2 is a cytosolic or periplasmic protein, we used two different treatments, lysozyme and freeze-thaw. The lysozyme treatment, a test for defining strict periplasmic localization, disrupts the cell's outer envelope, thus exposing the periplasmic space to the external environment. Freeze-thawing selectively releases cytosolic proteins attached to the inner surface of the cytoplasmic membrane into the periplasmic fraction (42). Cytosolic and periplasmic fractions obtained with freeze-thaw treatment (lanes 5 and 6, respectively) and cytosolic and periplasmic fractions obtained with lysozyme treatment (lanes 7 and 8, respectively) were subjected to immunoblotting analysis (Fig. 6A). Trx2 was identified in the same fraction as Trx1 in both treatments (lanes 6 and 7), suggesting not only its cytoplasmic localization but also its mainly peripheral association with the inner surface of the cytoplasmic membrane similarly to Trx1 (42). The identification of a novel thioredoxin sequence in the E. coli genome addresses the question of whether the protein coded by this sequence is active as thioredoxin and if it is normally expressed. We have tried to answer these questions and have demonstrated that this newly identified sequence codes for a protein with thioredoxin activity in the insulin, DTNB, and RNR assay. Also, this protein is expressed normally in E. coli cells as shown by Western blot analysis and activity assays. The existence of a second thioredoxin-like protein in E. coli has been proposed by Beckwith and co-workers (25, 26).
Thioredoxin reductase, the flavoenzyme that reduces thioredoxin via
NADPH, is implicated in the maintenance of the reducing environment of the E. coli cytoplasm, since all mutants selected to allow
disulfide bond formation in the cytoplasm mapped in the trxB
gene (25), which codes for thioredoxin reductase. Nevertheless,
E. coli thioredoxin reductase is a highly specific enzyme
only capable of reducing thioredoxin and not any other disulfide bonds
in cytoplasmic proteins. However, thioredoxin was not required for the
maintenance of the reducing environment, since the single mutant
trxA Reduction of ribonucleotides to their corresponding deoxyribonucleotides is an essential step in all living organisms in which the building blocks for DNA synthesis are supplied. RNR, the enzyme responsible for this reaction is thus tightly regulated, and the electron supply for its catalytic activity must be guaranteed. If we consider the recently described NrdH protein (see below) as an efficient electron donor for RNR (43), E. coli Trx2 is the fifth electron donor identified for RNR. Whether all of these proteins can act as electron donors for RNR under physiological conditions or whether some of them support this function only when others are absent is a matter for further study based on the characterization of the respective mutants. Several attempts in the past to identify any residual thioredoxin
activity by biochemical methods in E. coli mutants lacking Trx1 have failed (20, 26, 41, 44). By assaying heated extracts
(85 °C, 5 min) of K38 (wild type) and A179
(trxA The main difference between this novel Trx2 and the rest of the
prokaryotic thioredoxins is the existence of 32 extra amino acids
residues in its N terminus. This region of the protein does not match
with any consensus sequence for translocation in E. coli,
and the cytoplasmic localization of Trx2 was confirmed by Western blot
analysis of periplasmic and cytosolic fractions. The co-localization of
both thioredoxins suggests the attachment of Trx2 to the inner surface
of the cytoplasmic membrane as it has been described previously for
Trx1 (42). The levels of Trx2 were similar in the wild type and the
trxA Interestingly, the N-terminal domain of E. coli Trx2 is
homologous to the N terminus part of C. nephridii Trx3 (34).
C. nephridii Trx3 could complement some
thioredoxin-deficient E. coli mutant phenotypes (namely
growth in minimal medium with methionine sulfoxide as the only
methionine source and support of growth of bacteriophages T7 and M13
but not f1 growth), when cloned in a high expression vector.
Thioredoxin-1-deficient mutants of E. coli have several
phenotypes that should be expected to be complemented by the novel
Trx2. The maintenance of these phenotypes in the trxA mutant
indicates that either the function of Trx2 is different from that of
Trx1 or the molecule number per cell of Trx2 is not enough to
substitute Trx1. Expression of Trx2 from a high expression vector in a
trxA It is also interesting to point out that the disposition of the four cysteine residues in the N terminus of Trx2 resembles the structure of a zinc finger. Zinc finger motifs have been described in various DNA-binding proteins, and the importance of the cysteine residues is supported by the observation that modification of these residues inhibited enzymatic activity (46). E. coli primase is an example of a bacterial protein with only one zinc finger domain that binds specific sequences in the DNA (47). In fact, the zinc finger domain of primase is located in the N terminus of the protein, similar to E. coli Trx2. We are currently exploring the possibility that E. coli Trx2 might contain a zinc finger motif. The C-terminal part of the E. coli Trx2 is highly homologous to the rest of the prokaryotic thioredoxins with many amino acid residues conserved apart from those of the active site, including those necessary for protein-protein interactions or for maintenance of the native structure of the molecule. There is a Pro-108 at the position equivalent to Pro-76 in Trx1 that may serve to stabilize the active site. Ala-61, Trp-63, Asp-58, and Lys-89 (Ala-29, Trp-31, Asp-26, and Lys-57 in Trx1), which are invariant between thioredoxins, are also conserved in Trx2 (48). Prolines and glycines are common residues in or close to bends in proteins. Additionally, they are highly conserved in thioredoxins and fulfill this function (Gly-84, Gly-92, and Pro-64 in Trx1). There are also Gly residues in Trx2 (Gly-116, Gly-124); however, there is an Arg-96 at the corresponding position of Pro-64. Trx2 is an atypical thioredoxin with an N-terminal extension whose potential functions still represent an open question. Although the biological function of Trx2 remains unknown, maintenance of the reducing environment in cytoplasm is likely to be such a function. Determination of the redox potential of Trx2 as well as disruption of its gene are necessary to answer these questions. Whether Trx2 is represented in higher organisms than prokaryotes will also be of great interest. We finally propose the nomenclature trxC for the gene and Trx2 for the protein corresponding to this novel thioredoxin in E. coli. * This work was supported by grants from the Swedish Medical Research Council (Project 13X-10370) and Spanish Ministerio de Educación y Cultura (to A. M.-V.).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. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U85942.
These two authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biosciences, Center for Biotechnology, Karolinska Institutet, Novum, S-141 57 Huddinge, Sweden. Tel.: 46-8-6089162; Fax: 46-8-7745538; E-mail: Giannis.Spyrou{at}mbb.ki.se. 1 The abbreviations used are: Trx, thioredoxin; RNR, ribonucleotide reductase; Trx-S2, oxidized thioredoxin; Trx-(SH)2, reduced thioredoxin; Grx, glutaredoxin; DTT, dithiothreitol; DTNB, 5,5 -dithiobis-(2-nitrobenzoic acid); PCR, polymerase chain reaction; PEG, polyethylene glycol; BSA,
bovine serum albumin.
2 The sequence is available on the World Wide Web at the server for the E. coli data base collection (http://susi.bio.uni-giessen.de/ecdc.html). We thank Dr. Albert Jordan, Rolf Eliasson, and Prof. Britt-Marie Sjöberg for help with RNR determinations and RNR supply.
Volume 272, Number 49,
Issue of December 5, 1997
pp. 30841-30847
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A. Shahpiri, B. Svensson, and C. Finnie The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase Plant Physiology, February 1, 2008; 146(2): 789 - 799. [Abstract] [Full Text] [PDF] |
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J. Ye, S.-H. Cho, J. Fuselier, W. Li, J. Beckwith, and T. A. Rapoport Crystal Structure of an Unusual Thioredoxin Protein with a Zinc Finger Domain J. Biol. Chem., November 30, 2007; 282(48): 34945 - 34951. [Abstract] [Full Text] [PDF] |
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P. Vattanaviboon, W. Tanboon, and S. Mongkolsuk Physiological and Expression Analyses of Agrobacterium tumefaciens trxA, Encoding Thioredoxin J. Bacteriol., September 1, 2007; 189(17): 6477 - 6481. [Abstract] [Full Text] [PDF] |
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J. Beckwith What Lies Beyond Uranus?: Preconceptions, Ignorance, Serendipity and Suppressors in the Search for Biology's Secrets Genetics, June 1, 2007; 176(2): 733 - 740. [Full Text] [PDF] |
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T. Zeller, K. Li, and G. Klug Expression of the trxC Gene of Rhodobacter capsulatus: Response to Cellular Redox Status Is Mediated by the Transcriptional Regulator OxyR J. Bacteriol., November 1, 2006; 188(21): 7689 - 7695. [Abstract] [Full Text] [PDF] |
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A. P. Fernandes, M. Fladvad, C. Berndt, C. Andresen, C. H. Lillig, P. Neubauer, M. Sunnerhagen, A. Holmgren, and A. Vlamis-Gardikas A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase J. Biol. Chem., July 1, 2005; 280(26): 24544 - 24552. [Abstract] [Full Text] [PDF] |
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K. Li, C. Pasternak, E. Hartig, K. Haberzettl, A. Maxwell, and G. Klug Thioredoxin can influence gene expression by affecting gyrase activity Nucleic Acids Res., August 24, 2004; 32(15): 4563 - 4575. [Abstract] [Full Text] [PDF] |
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H. Schmidt and R. L. Krauth-Siegel Functional and Physicochemical Characterization of the Thioredoxin System in Trypanosoma brucei J. Biol. Chem., November 21, 2003; 278(47): 46329 - 46336. [Abstract] [Full Text] [PDF] |
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J.-F. Collet, J. C. D'Souza, U. Jakob, and J. C. A. Bardwell Thioredoxin 2, an Oxidative Stress-induced Protein, Contains a High Affinity Zinc Binding Site J. Biol. Chem., November 14, 2003; 278(46): 45325 - 45332. [Abstract] [Full Text] [PDF] |
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C. H. Lillig, A. Potamitou, J.-D. Schwenn, A. Vlamis-Gardikas, and A. Holmgren Redox Regulation of 3'-Phosphoadenylylsulfate Reductase from Escherichia coli by Glutathione and Glutaredoxins J. Biol. Chem., June 13, 2003; 278(25): 22325 - 22330. [Abstract] [Full Text] [PDF] |
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C. M. Sadek, A. Jimenez, A. E. Damdimopoulos, T. Kieselbach, M. Nord, J.-A. Gustafsson, G. Spyrou, E. C. Davis, R. Oko, F. A. van der Hoorn, et al. Characterization of Human Thioredoxin-like 2. A NOVEL MICROTUBULE-BINDING THIOREDOXIN EXPRESSED PREDOMINANTLY IN THE CILIA OF LUNG AIRWAY EPITHELIUM AND SPERMATID MANCHETTE AND AXONEME J. Biol. Chem., April 4, 2003; 278(15): 13133 - 13142. [Abstract] [Full Text] [PDF] |
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L. M. S. Baker and L. B. Poole Catalytic Mechanism of Thiol Peroxidase from Escherichia coli. SULFENIC ACID FORMATION AND OVEROXIDATION OF ESSENTIAL CYS61 J. Biol. Chem., March 7, 2003; 278(11): 9203 - 9211. [Abstract] [Full Text] [PDF] |
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K. Li, E. Hartig, and G. Klug Thioredoxin 2 is involved in oxidative stress defence and redox-dependent expression of photosynthesis genes in Rhodobacter capsulatus Microbiology, February 1, 2003; 149(2): 419 - 430. [Abstract] [Full Text] [PDF] |
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Y. Chen, J. Cai, T. J. Murphy, and D. P. Jones Overexpressed Human Mitochondrial Thioredoxin Confers Resistance to Oxidant-induced Apoptosis in Human Osteosarcoma Cells J. Biol. Chem., August 30, 2002; 277(36): 33242 - 33248. [Abstract] [Full Text] [PDF] |
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A. Potamitou, A. Holmgren, and A. Vlamis-Gardikas Protein Levels of Escherichia coli Thioredoxins and Glutaredoxins and Their Relation to Null Mutants, Growth Phase, and Function J. Biol. Chem., May 17, 2002; 277(21): 18561 - 18567. [Abstract] [Full Text] [PDF] |
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A. Vlamis-Gardikas, A. Potamitou, R. Zarivach, A. Hochman, and A. Holmgren Characterization of Escherichia coli Null Mutants for Glutaredoxin 2 J. Biol. Chem., March 22, 2002; 277(13): 10861 - 10868. [Abstract] [Full Text] [PDF] |
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L. M. S. Baker, A. Raudonikiene, P. S. Hoffman, and L. B. Poole Essential Thioredoxin-Dependent Peroxiredoxin System from Helicobacter pylori: Genetic and Kinetic Characterization J. Bacteriol., March 15, 2001; 183(6): 1961 - 1973. [Abstract] [Full Text] |
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M.-J. Prieto-Alamo, J. Jurado, R. Gallardo-Madueno, F. Monje-Casas, A. Holmgren, and C. Pueyo Transcriptional Regulation of Glutaredoxin and Thioredoxin Pathways and Related Enzymes in Response to Oxidative Stress J. Biol. Chem., April 28, 2000; 275(18): 13398 - 13405. [Abstract] [Full Text] [PDF] |
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W. K. Ray, G. Zeng, M. B. Potters, A. M. Mansuri, and T. J. Larson Characterization of a 12-Kilodalton Rhodanese Encoded by glpE of Escherichia coli and Its Interaction with Thioredoxin J. Bacteriol., April 15, 2000; 182(8): 2277 - 2284. [Abstract] [Full Text] |
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D. Ritz, H. Patel, B. Doan, M. Zheng, F. Aslund, G. Storz, and J. Beckwith Thioredoxin 2 Is Involved in the Oxidative Stress Response in Escherichia coli J. Biol. Chem., January 28, 2000; 275(4): 2505 - 2512. [Abstract] [Full Text] [PDF] |
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J. Shi, A. Vlamis-Gardikas, F. Aslund, A. Holmgren, and B. P. Rosen Reactivity of Glutaredoxins 1, 2, and 3 from Escherichia coli Shows That Glutaredoxin 2 Is the Primary Hydrogen Donor to ArsC-catalyzed Arsenate Reduction J. Biol. Chem., December 17, 1999; 274(51): 36039 - 36042. [Abstract] [Full Text] [PDF] |
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C. H. Lillig, A. Prior, J. D. Schwenn, F. Aslund, D. Ritz, A. Vlamis-Gardikas, and A. Holmgren New Thioredoxins and Glutaredoxins as Electron Donors of 3'-Phosphoadenylylsulfate Reductase J. Biol. Chem., March 19, 1999; 274(12): 7695 - 7698. [Abstract] [Full Text] [PDF] |
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J. R. Pedrajas, E. Kosmidou, A. Miranda-Vizuete, J.-A. Gustafsson, A. P. H. Wright, and G. Spyrou Identification and Functional Characterization of a Novel Mitochondrial Thioredoxin System in Saccharomyces cerevisiae J. Biol. Chem., March 5, 1999; 274(10): 6366 - 6373. [Abstract] [Full Text] [PDF] |
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F. Åslund and J. Beckwith The Thioredoxin Superfamily: Redundancy, Specificity, and Gray-Area Genomics J. Bacteriol., March 1, 1999; 181(5): 1375 - 1379. [Full Text] |
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B. J. Laughner, P. C. Sehnke, and R. J. Ferl A Novel Nuclear Member of the Thioredoxin Superfamily Plant Physiology, November 1, 1998; 118(3): 987 - 996. [Abstract] [Full Text] |
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M. K. B. Berlyn Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984. [Abstract] [Full Text] [PDF] |
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L. Debarbieux and J. Beckwith The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm PNAS, September 1, 1998; 95(18): 10751 - 10756. [Abstract] [Full Text] [PDF] |
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M. Dormeyer, N. Reckenfelderbaumer, H. Ludemann, and R. L. Krauth-Siegel Trypanothione-dependent Synthesis of Deoxyribonucleotides by Trypanosoma brucei Ribonucleotide Reductase J. Biol. Chem., March 30, 2001; 276(14): 10602 - 10606. [Abstract] [Full Text] [PDF] |
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F. Monje-Casas, J. Jurado, M.-J. Prieto-Alamo, A. Holmgren, and C. Pueyo Expression Analysis of the nrdHIEF Operon from Escherichia coli. CONDITIONS THAT TRIGGER THE TRANSCRIPT LEVEL IN VIVO J. Biol. Chem., May 18, 2001; 276(21): 18031 - 18037. [Abstract] [Full Text] [PDF] |
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