Báo cáo Y học: Identification and characterization of thioredoxin and thioredoxin reductase fromAeropyrum pernixK1 pdf

Identification and characterization of thioredoxin and thioredoxin
reductase from
Aeropyrum pernix
K1
Sung-Jong Jeon and Kazuhiko Ishikawa
National Institute of Advanced Industrial Science and Technology (Kansai), Ikeda, Osaka, Japan
We have identified and characterized a thermostable thio-
redoxin system in the aerobic hyperthermophilic archaeon
Aeropyrum pernix K1. The gene (Accession no. APE0641) of
A. pernix encoding a 37 kDa protein contains a redox active
site motif (CPHC) but its N-terminal extension region
(about 200 residues) shows no homology within the genome
database. A second gene (Accession no. APE1061) has high
homology to thioredoxin reductase and encodes a 37 kDa
protein with the active site motif (CSVC), and binding sites
forFADandNADPH.Weclonedthetwogenesand
expressed both proteins in E. coli. It was observed that the
recombinant proteins could act as an NADPH-dependent
protein disulfide reductase system in the insulin reduction. In
addition, the APE0641 protein and thioredoxin reductase
from E. coli could also catalyze the disulfide reduction.
These indicated that APE1061 and APE0641 express thio-
redoxin (ApTrx) and thioredoxin reductase (ApTR) of
A. pernix, respectively. ApTR is expressed as an active
homodimeric flavoprotein in the E. coli system. The opti-
mum temperature was above 90 °C, and the half-life of heat
inactivation was about 4 min at 110 °C. The heat stability of
ApTR was enhanced in the presence of excess FAD. ApTR
could reduce both thioredoxins from A. pernix and E. coli
and showed a similar molar specific activity for both pro-
teins. The standard state redox potential of ApTrx was about

)262 mV, which was slightly higher than that of Trx from
E. coli ()270 mV). These results indicate that a lower redox
potential of thioredoxin is not necessary for keeping catalytic
disulfide bonds reduced and thereby coping with oxidative
stress in an aerobic hyperthermophilic archaea. Further-
more, the thioredoxin system of aerobic hyperthermophilic
archaea is biochemically close to that of the bacteria.
Keywords: thioredoxin; thioredoxin reductase; hyper-
thermophile; aerobic archaea; Aeropyrum pernix.
The thioredoxin system composed of thioredoxin (Trx),
thioredoxin reductase (TR), and NADPH serves as a
hydrogen donor system for specific reduction of disulfide
bonds in proteins [1,2]. Trxs are small monomeric proteins
with a typical CXXC active site motif that catalyzes many
redox reactions through thiol-disulfide exchange. Oxidized
Trx (Trx-S
2
) can be reduced by NADPH and the flavo-
enzyme TR. This reduced Trx (Trx-(SH)
2
) is able to catalyze
the reduction of disulfides in a number of proteins (Reaction
1and2).
Trx-S
2
þ NADPH þ H
þ
ÀÀ*
)ÀÀ
TR

Trx-(SH)
2
þ NADP
þ
Reaction 1
Trx-(SH)
2
þ Protein-S
2
ÀÀ*
)ÀÀ Trx-S
2
þ Protein-(SH)
2
Reaction 2
Since the discovery of the first Escherichia coli Trx, which
was shown to act as an electron donor for ribonucleotide
reductase and therefore essential for DNA synthesis [3], Trx
has been isolated and characterized from bacteria, eukar-
yotes and the anaerobic archaeon Methanococcus jannaschii
[4]. Trx can function as an electron donor for ribonucleotide
reductase, 3¢-phosphoadenosine-5¢-phosphosulfate reduc-
tase, and methionine-sulfoxide reductase in bacterial and
eukaryotic cells [5,6]. In addition, it has been shown that
Trxs are involved in the activation of DNA-binding activity
of transcription factors [7].
Thioredoxin reductase (TR) is a homodimeric flavoen-
zyme containing a redox-active disulfide and a FAD in each
subunit [8]. The enzymatic mechanism of TR involves the
transfer of reducing equivalents from NADPH to a redox-

active disulfide via FAD [9]. On the basis of the differences
in size, structure and catalytic mechanism, two classes of TR
can be distinguished [10]. The low molecular mass proteins
from E. coli [11] and Saccharomyces cerevisiae [12] are
dimers of 35 kDa subunits, whereas the high molecular
mass proteins from higher eukaryotes, including mammals
[13,14], Caenorhabditis elegans [15] and Plasmodium falci-
parum [16] are dimers of 55 to 58 kDa subunits. TRs of both
classes are members of a larger family of pyridine nucleotide
disulfide oxidoreductases that includes lipoamide dehydrog-
enase, glutathione reductase and mercuric reductase [17].
Bacterial TR is distinct from those of mammalian origin.
The bacterial enzyme is highly specific for the homologous
Trx as a substrate [18]. In contrast, mammalian TR has a
broader substrate specificity and can reduce not only thiore-
doxins from different species but also many nondisulfides,
Correspondence to K. Ishikawa, The special division for Human Life
Technology, National Institute of Advanced Industrial Science and
Technology (Kansai), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577,
Japan. Fax: + 81 727 51 9628, Tel.: + 81 727 51 9526,
E-mail:
Abbreviations: GSH, reduced glutathione; GSSG, glutathione
disulfide (oxidized); HED, b–hydroxyethyl disulfide; IPTG, isopropyl
thio-b-
D
-galactoside; NBS
2
,5,5¢-dithiobis(2-nitrobenzoic acid);
Trx, thioredoxin; TR, thioredoxin reductase.
Enzyme: thioredoxin reductase (E.C. 1.6.4.5).

(Received 11 July 2002, revised 16 August 2002,
accepted 5 September 2002)
Eur. J. Biochem. 269, 5423–5430 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03231.x
such as 5,5¢-dithiobis(2-nitrobenzoic acid) (NBS
2
), selenite,
selenoglutathione, vitamin K and alloxan [9,19]. In addition
to TR having been isolated and characterized from a wide
variety of bacterial and eukaryotic species, it has also been
found in anaerobic hyperthermophilic archaea.
One of the most important functions of Trx is the
reduction of reactive oxygen species, which is performed by
the interaction of thioredoxin and thioredoxin peroxidases
[20]. Therefore, the study of the thioredoxin system in
aerobic hyperthermophilic archaea should be informative.
There is no study in the literature about TR from aerobic
hyperthermophilic archaea. In the genome database of the
aerobic hyperthermophilic archaeon Aeropyrum pernix K1,
we found a new gene which codes for a 37 kDa protein with
a redox-active site motif (CPHC). The protein is about three
times as large as the normal Trx.
To understand the role of the gene and Trx/TR system in
aerobic hyperthermophilic archaea, we have cloned two
genes, the first encoding a protein (APE0641) containing a
redox active site motif (CPHC) and the second a TR
homologue protein (APE1061) from A. pernix,whichwere
then characterized. In this paper, we study the Trx/TR
system of the aerobic archaea.
MATERIALS AND METHODS
Materials

The plasmid pET-3d was purchased from Novagen
(Madison, WI, USA). KOD DNA polymerase and T
4
DNA polymerase were purchased from Toyobo (Osaka,
Japan). NADPH and NADP were obtained from Oriental
Yeast (Tokyo, Japan). Bovine insulin, glutathione reduc-
tase, E. coli thioredoxin, and E. coli thioredoxin reductase
were obtained from Sigma (St. Louis, MO, USA).
Glutathione (oxidized form, GSSG), 5,5¢-dithiobis(2-nitro-
benzoic acid) (NBS
2
), and b-hydroxyethyl disulfide (HED)
were obtained from Wako Pure Chemical Industries
(Tokyo, Japan). Other reagents were of the reagent grade
available.
Cloning and expression of
A. pernix TRX
and
TR
Chromosomal DNA of A. pernix K1 was prepared as
described by Sako et al. [21]. The gene (APE0641) was
amplified by PCR using chromosomal DNA as a template,
and the two primers TX1: 5¢-ATGGTCGCGTCGACC
TTCGTAGTA-3¢ (forward); and TX2: 5¢-
GGATCCTCA
GCCCCCGTATATCTCCCT-3¢ (reverse), which were
designed based on an open reading frame coding for a
protein of 349 amino acids. Amplification was carried out at
94 °C for 30 s, 55 °Cfor2s,74°C for 30 s for 30 cycles
using KOD DNA polymerase. The plasmid pET-3d was

then digested with NcoI, treated with T
4
DNA polymerase
to fill in the cohesive ends and digested with BamHI again.
The amplified PCR product was digested with BamHI (the
BamHI site in primer TX2 is underlined) and inserted into
the pET-3d vector. The recombinant plasmid was designa-
ted pTRX. Confirmation of the gene sequence in pET-3d
was carried out by DNA sequencing, using the ABI Prism
310 genetic analyzer of Applied Biosystems (Foster city,
CA, USA). E. coli BL21(DE3) cells were transformed with
pTRX and incubated in NZCYM medium containing
ampicilin (100 lgÆmL
)1
)at37°C until the optical density at
600 nm reached 0.5. Expression was induced by the
addition of 0.5 m
M
isopropyl thio-b-
D
-galactoside and cells
were incubated further for 4 h at 37 °C.
APE1061 was amplified by PCR using the chromosomal
DNA as template, and two primers, TR1, 5¢-ATTAGG
TGCGTGATTATGCCG-3¢ as the forward primer and
TR2, 5¢-GGATCCTTACTTTAACCCAGTTAAAGG-3¢
as the reverse primer. The amplified fragment was inserted
into the pET-3d vector, and the resulting plasmid was
designated pTR. The methods for cloning and overexpres-
sion of the gene were identical to those described above.

Purification of the recombinant proteins
The recombinant proteins from APE0641 and APE1061
were expressed and prepared from E. coli BL21(DE3)/
pTRX and Rosetta (DE3)/pTR, respectively. One-litre
cultures of cells harboring pTRX were harvested by
centrifugation at 7000 g for 10 min and frozen at )70 °C.
The thawed cells were then disrupted by sonication in
40 mL of buffer A (50 m
M
Tris/HCl, pH 8.0, 0.1 m
M
EDTA). The suspension of disrupted cells was centrifuged
at 27 000 g for 30 min and the supernatant fraction was
heat-treated at 80 °C for 30 min followed by recentrifuga-
tion. The supernatant was loaded on a HiTrap Q column
from Amersham Biosciences (Piscataway, NJ, USA),
equilibrated in buffer A and the bound protein was eluted
with a linear gradient of NaCl (0–1.0
M
in the same buffer).
The protein solution was concentrated using a centricon 10
filter from Amicon (Millipore, Bedford, MA, USA) and
dialyzed against buffer B (50 m
M
sodium phosphate,
pH 7.0, 150 m
M
NaCl). The dialyzed solution was loaded
on a HiPrep Sephacryl S-200 HR 26/60 column (Amersham
Biosciences) and eluted with buffer B. In the case of

recombinant protein from APE1061, after application of a
HiTrap Q column, the fractions containing thioredoxin
reductase activity were pooled, dialyzed against buffer A,
and applied to a HiTrap Blue column (Amersham Bio-
sciences), and the recombinant protein was eluted with 2
M
NaCl. Purity of the recombinant protein was assayed by
0.1–12% SDS/PAGE. Protein concentration was deter-
mined using protein assay system of Bio-Rad (Hercules,
CA, USA) with BSA as the standard.
Molecular mass determination
The molecular masses of the recombinant proteins were
determined by SDS/PAGE and gel filtration on a Sephacryl
S-200 HR 26/60 column (Amersham Biosciences) equili-
brated with buffer B at a flow rate of 2 mLÆmin
)1
.
FAD contents of the recombinant protein
The quantitative extraction of FAD from the recombinant
protein was achieved by incubation in a sealed tube at
110 °C for 30 min. After the incubation, the denatured and
precipitated protein was removed by centrifugation [22].
The concentration of free flavin was determined from the
absorbance at 450 nm with a molar extinction coefficient of
11.3 m
M
)1
Æcm
)1
.

5424 S J. Jeon and K. Ishikawa (Eur. J. Biochem. 269) Ó FEBS 2002
Thioredoxin activity assays
Thioredoxin activity was determined by the insulin preci-
pitation assay described by Holmgren [23]. The standard
assay mixture contained 0.1
M
potassium phosphate
(pH 7.0), 1 m
M
EDTA, and 0.13 m
M
bovine insulin in the
absence or in the presence of the recombinant protein, and
the reaction was initiated upon the addition of 1 m
M
dithiothreitol. An increase of the absorbance at 650 nm
was monitored at 30 °C. The thioredoxin activity with
thioredoxin reductase was assayed by use of the insulin
reduction assay as described elsewhere [24]. Aliquots of
thioredoxin were preincubated at 37 °Cfor20minwith
2 lLof50m
M
Hepes, pH 7.6, 100 lgÆmL BSA and 2 m
M
dithiothreitol in a total volume of 50 lL. Then, 40 lLofa
reaction mixture composed of 200 lLofHepes(1
M
),
pH 7.6, 40 lLofEDTA(0.2
M

), 40 lLofNADPH
(40 mgÆmL
)1
), and 500 lL of insulin (10 mgÆmL
)1
)were
added. The reaction started with the addition of 10 lLof
thioredoxin reductase, and incubation was continued for
20 min at 37 °C. The reaction was stopped by the addition
of 0.5 mL of 6
M
guanidine-HCl and 1 m
M
NBS
2
,andthe
absorbance at 412 nm was measured. Glutaredoxin activity
was measured using the glutathione-disulfide transhydro-
genase assay described by Gan et al.[25].
Thioredoxin reductase activity assays
Assays for thioredoxin reductase activity were carried out by
two methods at 30 °C. In the NBS
2
reduction assay [26], the
purified thioredoxin reductase (50 n
M
) was added to the
reaction mixtures containing 5–400 l
M
NBS

2
and 0.2 m
M
NADPH in assay buffer (100 m
M
potassium phosphate,
pH 7.0, 2 m
M
EDTA), and activity was calculated from the
increase in absorbance at 412 nm using a molar extinction
coefficient of 27.2 m
M
)1
Æcm
)1
, since reduction of DTNB by
1 mol of NADPH yields 2 mol of 2-nitro-5-thiobenzoate
(e
412
¼ 13.6 m
M
)1
Æcm
)1
). In the thioredoxin reduction assay
[26], enzymes were added to the reaction mixtures containing
0.2–4 l
M
ApTrx, 0.2 m
M

NADPH, and 0.5 mgÆmL
)1
insu-
lin in assay buffer, and activity was calculated from the
decrease in absorbance at 340 nm using a molar extinction
coefficient of 6.22 m
M
)1
Æcm
)1
.TheK
m
and V
max
values were
obtained by the damped nonlinear least-squares method
(Marquardt–Levenberg method) [27,28].
Redox potential of thioredoxin
The reversibility of the reaction NADPH + TrxS
2
+
H
+
« Trx(SH)
2
+NADP
+
was employed for determin-
ing the equilibrium constant using the absorbance change at
340 nm. Thioredoxin (5–40 l

M
)wasmixedwith50l
M
NADPH in a total volume of 500 lLatpH7.0,25°C,
followed by addition of 50 n
M
thioredoxin reductase and
then excess NADP
+
(1200 l
M
) as described previously [29].
From the equilibrium concentrations, redox potentials were
calculated according to the Nernst equation:
E
o
¢(substrate) ¼ E
o
¢(NADP
+
)+(RT/nF )
· ln([NADP
+
][substrate
red
]/[NADPH][substrate
ox
])
A value of )0.315 V was used as the redox potential of
NADP

+
[30].
RESULTS
Cloning of the two genes from
A. pernix
K1
In the A. pernix K1 genome database (.
nite.go.jp/cgi-bin/dogan/genome_top.cgi?ÔapeÕ), we identi-
fied an ORF (Accession no. APE0641) encoding a protein
that contained the CXXC motif and an ORF (APE1061)
encoding a thioredoxin reductase homologue. Both ORFs
were amplified by PCR, cloned and sequenced to confirm
the sequences described in the genome database.
The APE0641 gene encodes a protein of 349 amino acids
with a predicted molecular mass of 37082 Da. Within the
C-terminal region, the deduced amino acid sequence shows
a 23% identity (51% similar) with Trx of Saccharomyces
cerevisiae andislesssimilartothatofE. coli (Fig. 1). This
protein is larger than the classical thioredoxins in size, and
has a CPHC sequence that is different from the other
thioredoxins. In addition, it has two extra cysteine residues
at positions 140 and 216. APE1061 encodes a protein of 343
amino acids with a predicted molecular mass of 37 157 Da,
containing the redox active site (CSVC).
Molecular properties of the recombinant proteins
The proteins from APE0641 and APE1061 were expressed
in E. coli cells, and the recombinant proteins were purified
to homogeneity. Expression and subsequent purification
yielded 2.4 mg and 0.9 mg from a 1-L culture for APE0641
and APE1061, respectively. The molecular masses of the

proteins from APE0641 and APE1061 were estimated to be
about 37 and 36.5 kDa by SDS/PAGE, respectively
(Fig. 2A). These values are in agreement with the values
deduced by the gene sequence analysis. The native
molecular mass of the TR-like protein (ApTR) from
APE1061 was determined to be about 75 kDa by gel
filtration with a Sephacryl S-200 HR 26/60 (Fig. 2B). The
results suggested that the native state of ApTR is homo-
dimeric, similar to TR of E. coli [31]. The purified ApTR
Fig. 1. Alignment of Ap Trx with other classical thioredoxins. The redox
active site is enclosed in a rectangle. Asterisks indicate conserved amino
acid residues among three thioredoxins, A. pernix, Aeropyrum pernix;
S. cerevi, Saccharomyces cerevisiae; E. coli, Escherichia coli.
Ó FEBS 2002 Thioredoxin system of Aeropyrum pernix (Eur. J. Biochem. 269) 5425
showed a visible absorption spectrum typical for flavopro-
teins with absorbance maxima at 380 and 460 nm (Fig. 3).
The ratio between A
280
and A
460
for the ApTR is 8.1, in
agreement with 8.0 of the rat liver protein [32]. After the
addition of 5 molar equiv of NADPH, the enzyme is
reduced and the visible part of the spectrum is bleached
(Fig. 3). In addition, the fluorescence spectrum showed a
maximum at about 520 nm as observed for E. coli TR
[33,34] (Fig. 3). Recombinant protein for APE0641 exhi-
bited only the absorbance maximum at 280 nm.
Activity of thioredoxin
Thioredoxins are known to possess an activity as disulfide

reductases of insulin [4]. Reduction of insulin disulfide
bonds can be measured by the increase in turbidity due to
precipitation of the free insulin B-chain [23]. The reduction
of insulin by dithiothreitol was followed at 30 °Cand
pH 7.0. We compared the activities of E. coli Trx with the
recombinant protein from APE0641. The recombinant
protein had an activity of disulfide reductase with insulin
and showed threefold lower molar specific activity than that
of E. coli Trx (Fig. 4). We also examined whether the
recombinant protein has insulin reductase activity with
NADPH in the presence of A. pernix and E. coli TR. The
result that the insulin disulfide bonds were reduced in
the presence of the recombinant protein indicates that the
recombinant protein from APE0641 is a thioredoxin from
A. pernix (ApTrx) and that both proteins constitute a
thioredoxin system of A. pernix (Fig. 5A). Furthermore,
ApTrx catalyzed the disulfide reduction of insulin with the
E. coli TR (Fig. 5B). This phenomenon is not observed for
the other enzymes. Although ApTrx is not homologous to
the E. coli Trx, the result indicates that it can serve as
substrate for the E. coli TR. In addition, ApTR can reduce
Trxs from A. pernix and E. coli and show a similar molar
specific activity for both proteins (Fig. 5A). To understand
the function of two extra cysteines present at positions 140
and 216 of ApTrx, we preincubated ApTrx with a reducing
compound such as dithiothreitol. The dithiothreitol-
preincubated ApTrx showed a similar activity to the
nonpreincubated one, assayed with both A. pernix and
E. coli thioredoxin reductases (Fig. 5A,B). These results
indicate that ApTrx contains two extra cysteines but its

activity is not affected by dithiothreitol, suggesting that the
additional cysteine residues were not involved in regulating
its enzymatic activity [18,24,35]. We also confirmed that
Fig. 3. Spectroscopic properties of the recombinant ApTR. Absorption
spectra of a 12-l
M
enzyme in 50 m
M
potassium phosphate buffer,
pH 7.0, and 0.5 m
M
EDTA (solid line) and of the reduced enzyme
after addition of 60 l
M
NADPH (dashed line). Fluorescence spectra of
ApTR recorded using a Hitachi F-4500 fluorescence spectrophoto-
meter by exciting at 380 nm (dotted line).
Fig. 2. SDS/PAGE and gel filtration analysis of ApTrx and ApTR. (A) The purified ApTrx and ApTR were subjected to SDS/PAGE on 0.1% SDS-
12% PAGE and stained with Coomassie Brilliant Blue R-250. Lane 1, Low molecular weight markers: phosphorylase b (94 kDa), albumin
(67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa); Lane 2, ApTrx; Lane 3, ApTR. (B) Molecular mass
determination of ApTrx and ApTR. Molecular masses of recombinant ApTrx and ApTR were determined by analysis of the elution files of standard
proteins from a Sephacryl S-200 HR 26/60 column. The column was calibrated with molecular mass standards from Amersham Biosciences:
catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), Ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), ribonuclease A (13.7 kDa).
5426 S J. Jeon and K. Ishikawa (Eur. J. Biochem. 269) Ó FEBS 2002
ApTrx had no thiol-transferase activity by a glutathione-
disulfide oxidoreductase (data not shown).
Redox potential of
Ap
Trx
The thioredoxin reductase reaction (Reaction 1) was fully

reversible by addition of excess NADP
+
with thioredoxin.
The time course of reduction and reoxidation for the
disulfide of ApTrx in the presence of 50 n
M
ApTR was
plotted in Fig. 6. The redox potential was determined from
the equilibrium constants according to the Nernst equation.
The A. pernix Trx(SH)
2
/TrxS
2
redox pair has a redox
potential of )262 mV at pH 7.0 and 25 °C, and shows a
higher redox potential than the value reported for E. coli
Trx ()270 mV) [29]. In the insulin reduction, the lower
activity of ApTrx is consistent with the higher redox
potential of ApTrx compared to that of E. coli Trx. The
redox potential of the E. coli cytosol has been estimated to
be approximately )260 to )280 mV [36], and the standard
state redox potential of ApTrx is contained within this
range.
Catalytic properties of
Ap
TR
To obtain the kinetic parameters of ApTR for various
substrates, we used the Trx and NBS
2
reduction assay as

described in Materials and methods. The kinetic parameters
of the reaction for ApTrx, NBS
2
,andNADPHare
summarized in Table 1. The K
m
of ApTR for recombinant
ApTrx at pH 7.0 and 25 °C was 12.3 ± 2.7 l
M
.TheK
m
of
ApTRforNADPHwas3.6±0.5l
M
and showed a
slightly lower K
m
value than that for the E. coli TR
(4.55 l
M
) [37]. Thus, ApTR catalyzed NADPH-dependent
reductions of ApTrx and NBS
2
, but it was not able to
catalyze NADPH-dependent reduction of GSSG and HED,
electron acceptors of glutathione reductase and glutaredoxin
activity, respectively.
Fig. 6. Determination of the redox potential of ApTrx. Reduction of
disulfide in 36.5 l
M

ApTrx was started by the addition of 50 n
M
ApTR. When the reaction had stopped, NADP
+
was added to a final
concentration of 1.2 m
M
.TheformationofNADP
+
and NADPH
was followed from the decrease and increase at 340 nm, respectively.
Fig. 5. Trx activity in thioredoxin systems of
A. pernix and E. coli. The assays were per-
formed with a 50 n
M
concentration of
A. pernix TR (A) and E. coli TR (B). s,
ApTrx; h, E. coli Trx. The filled symbols
show the assays with ApTrx preincubated with
dithiothreitol for 20 min at 37 °C.
Fig. 4. Reduction of insulin catalyzed by thioredoxin from A. pernix and
E. coli. The dithiothreitol-dependent reduction of bovine insulin
disulfide was carried out as described in Materials and methods. The
increase in turbidity at 650 nm is plotted against the reaction time. d,
negative control; h,1l
M
and n,2l
M
A. pernix Trx; j,1l
M

and m,
2 l
M
E. coli Trx.
Ó FEBS 2002 Thioredoxin system of Aeropyrum pernix (Eur. J. Biochem. 269) 5427
Thermophilicity and thermostability of
Ap
TR
The thermophilicity of ApTR was investigated by measur-
ing the NBS
2
reductase activity at increasing temperatures.
As indicated in Fig. 7A, ApTR showed the maximum
activity at 90 °C, which is in the temperature range for
growth of A. pernix [21]. The thermostability of the
flavoenzyme ApTR was estimated by measuring the residual
NBS
2
reductase activity at 30 °C after heat treatment at two
different temperatures in the presence or absence of FAD
(Fig. 7B). The recombinant ApTR showed high thermosta-
bility, and the half-life of heat inactivation was about 4 min
at 110 °C. Furthermore, the heat stability of ApTR was
enhanced by the addition of FAD to the incubation
mixture, similar to that previously reported for the flavo-
enzyme of Thermus aquaticus [38]. It has been shown that
the inactivation of flavoenzyme at high temperature is
caused by the dissociation of flavin from the enzyme and the
subsequent denaturation of the apoenzyme [38].
DISCUSSION

The ApTRX and ApTR genes for the thioredoxin system
have been cloned from A. pernix K1. Their recombinant
proteins were overexpressed and characterized biochemi-
cally.
The active site motif, CPHC, which is involved in ApTrx
isthesameasthatofE. coli DsbA, the most powerful
oxidant among thiol-disulfide oxidoreductases [39]. The two
central residues within the active site motif play a critical
role in determining the redox potential [40]. Nevertheless,
the redox potential of ApTrx is )262 mV, which is very
different from that of E. coli DsbA ()125 mV). This
indicates that amino acids other than those within the
active site are also important in determining redox potential
[41]. In an anaerobic hyperthermophile, it was suggested
that the lower redox potential is necessary to keep catalytic
disulfide bonds reduced and to cope with oxidative stress [4].
In an aerobic hyperthermophilic archaeon, however, the
result that the standard state redox potential of ApTrx was
slightly higher than that of Trx from E. coli ()270 mV)
indicates that the low redox potential of thioredoxin is not
necessary for these two processes. The redox potential of
Trx may also represent the environment that microorgan-
isms inhabit. The redox potential of ApTrx suggests that this
microorganism does not need more reduced environments
than those of E. coli. The molecule of ApTrxislargerthan
the other thioredoxin homologues in size due to an extended
region at the N-terminus. This region showed no homology
to sequences in the databases and the function is unknown.
However, ApTrx protein exhibits biochemical activities
similar to classical thioredoxin. Mammalian thioredoxins

have at least two extra cysteine residues that can undergo
oxidation, leading to inactivation by dimerization [9].
Inactivated mammalian TRX1 can be reactivated after
preincubation with dithiothreitol [9,35]. ApTrx contains two
extra cysteine residues, but its activity is not affected by
preincubation with dithiothreitol, indicating that the activity
of ApTrx is not dependent on the redox state of the protein.
In the present study, we have demonstrated the in vitro
biochemical activities for a novel member of the thioredoxin
family.
ApTR is phylogenetically closer to the bacterial than
mammalian TRs. The deduced amino acid sequence is most
homologous to TR-like protein (54% identity and 73%
similarity) from Sulfolobus solfataricus and shows a relat-
ively high homology to the TR of E. coli (34% identity and
54% similarity). This protein possesses three conserved
motifs responsible for binding of FAD near the N-terminus
(GXGXX [G/A]) and the C-terminus (GXFAAGD) and
NADPH near the middle of the protein (GGGXXA) in
addition to a redox active center (CSVC). Its subunit
molecular mass is similar to that of the E. coli TR (35 kDa)
and therefore belongs to the low M
r
class. NBS
2
is not
directly reduced by low M
r
thioredoxin reductase, but
instead requires the presence of thioredoxin as a redox

mediator [33]. However, it can be reduced directly by ApTR,
indicating that it has the broader substrate specificity than
that of low M
r
TRs. Typically, enzymes of this family
contain two identical subunits, each subunit containing one
redox active disulfide, one mole of FAD per subunit, and
Fig. 7. Thermophilicity and thermostability of
ApTR. (A) NBS
2
reductase activity of ApTR
was determined at the indicated temper-
atures, as described in Materials and methods.
Negative control reactions in the absence
oftheenzymewereperformedinparallel.(B)
Enzyme (1 l
M
ApTR in 50 m
M
potassium
phosphate, pH 7.0, 0.5 m
M
EDTA) was
incubated at 105 °C in the presence (s)or
absence (h) of FAD and at 110 °Cinthe
presence (d) or absence (j)ofFAD,andthe
residual NBS
2
reductase activity of samples
were measured at 30 °C.

Table 1. Kinetic parameters for Ap TR catalytic activities. The kinetic
parameters were determined as described in Materials and methods
using nonlinear least-squares method (27). The K
m
value for NADPH
was determined at 2–80 l
M
NADPH and 2 m
M
NBS
2
.Datarepresent
the mean (±SE) of three separate experiments.
K
m
(l
M
) k
cat
(S
)1
)
Trx 12.3 ± 2.7 63.2 ± 12.1
NBS
2
172.4 ± 35.8 9.0 ± 1.5
NADPH 3.6 ± 0.5 –
5428 S J. Jeon and K. Ishikawa (Eur. J. Biochem. 269) Ó FEBS 2002
conserved FAD and NADPH binding motifs. Indeed, the
recombinant ApTR is expressed as a homodimeric flavoen-

zyme in E. coli, as deduced from UV and fluorescence
spectra (Fig. 3). The flavin in the supernatant after heat
denaturation of the apoenzyme is shown to be FAD.
The FAD content of the flavoenzyme is obtained from the
absorption coefficient at 450 nm [38]. FAD content of the
purified ApTR is 0.54 mol FAD per mol of subunit. It is
suggested that the flavin is weakly bound to the apoprotein
and partly lost during enzyme isolation. ApTR is stable at
high temperature (Fig. 7B). The enzyme has 70% residual
activity even after a 60-minute incubation at 100 °C (data not
shown). We also have shown that heat stability of ApTR is
enhanced in the presence of an excess FAD. Subsequent
studies for flavin will be necessary to understand the catalytic
mechanism and thermostability of ApTR protein.
This is the first report to characterize a functional
thioredoxin system in aerobic hyperthermophilic archaea.
The thioredoxin system plays a critical role in redox
control by regulating the activity of an enzyme responsible
for transcription factors, DNA synthesis and antioxidant.
We have identified two archaeal genes that code for
this thioredoxin system and shown that two proteins
operate as a NADPH-dependent protein-disulfide reduc-
tase system. This thioredoxin system may play an
important role in controlling the redox status of aerobic
archaeal proteins.
ACKNOWLEDGEMENTS
S J. Jeon was supported by the New Energy Industrial Technology
Development Organization (NEDO).
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