APE1061 has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif CSVC, and binding sites for FAD and NADPH.. It was observed that the recombinan
Trang 1Identification and characterization of thioredoxin and thioredoxin
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 pernixK1 The gene (Accession no APE0641) of
A pernixencoding 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
for FAD and NADPH We cloned the two genes and
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 thiothio-redoxin 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 90C, and the half-life of heat inactivation was about 4 min at 110C 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()270mV) 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-S2) 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
1 and 2)
Trx-S2 þ NADPH þ Hþ*)TRTrx-(SH)2þ NADPþ
Reaction 1
Trx-(SH)2 þ Protein-S2*) Trx-S2þ 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: kazu-ishikawa@aist.go.jp
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)
Trang 2such as 5,5¢-dithiobis(2-nitrobenzoic acid) (NBS2), 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, which were
then characterized In this paper, we study the Trx/TR
system of the aerobic archaea
M A T E R I A L S A N D M E T H O D S
Materials
The plasmid pET-3d was purchased from Novagen
(Madison, WI, USA) KOD DNA polymerase and T4
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) (NBS2), 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 ofA 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
94C for 30s, 55 C for 2 s, 74 C for 30s for 30cycles
using KOD DNA polymerase The plasmid pET-3d was
then digested with NcoI, treated with T4DNA 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
310genetic 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) at 37C until the optical density at
600 nm reached 0.5 Expression was induced by the addition of 0.5 mMisopropyl thio-b-D-galactoside and cells were incubated further for 4 h at 37C
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 10min and frozen at)70 C The thawed cells were then disrupted by sonication in 40mL of buffer A (50mM Tris/HCl, pH 8.0, 0.1 mM EDTA) The suspension of disrupted cells was centrifuged
at 27 000 g for 30min and the supernatant fraction was heat-treated at 80C for 30min 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 (0–1.0Min the same buffer) The protein solution was concentrated using a centricon 10 filter from Amicon (Millipore, Bedford, MA, USA) and dialyzed against buffer B (50mM sodium phosphate,
pH 7.0, 150 mMNaCl) 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 2M 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
110C for 30min After the incubation, the denatured and precipitated protein was removed by centrifugation [22] The concentration of free flavin was determined from the absorbance at 450nm with a molar extinction coefficient of 11.3 mM )1Æcm)1
Trang 3Thioredoxin activity assays
Thioredoxin activity was determined by the insulin
preci-pitation assay described by Holmgren [23] The standard
assay mixture contained 0.1M potassium phosphate
(pH 7.0), 1 mMEDTA, and 0.13 mMbovine insulin in the
absence or in the presence of the recombinant protein, and
the reaction was initiated upon the addition of 1 mM
dithiothreitol An increase of the absorbance at 650nm
was monitored at 30C 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 37C for 20 min with
2 lL of 50 mMHepes, pH 7.6, 100 lgÆmL BSA and 2 mM
dithiothreitol in a total volume of 50 lL Then, 40 lL of a
reaction mixture composed of 200 lL of Hepes (1M),
pH 7.6, 40 lL of EDTA (0 2M), 40 lL of NADPH
(40mgÆmL)1), and 500 lL of insulin (10mgÆmL)1) were
added The reaction started with the addition of 10 lL of
thioredoxin reductase, and incubation was continued for
20min at 37C The reaction was stopped by the addition
of 0.5 mL of 6Mguanidine-HCl and 1 mMNBS2, and the
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 30C In the NBS2reduction assay [26], the
purified thioredoxin reductase (50nM) was added to the
reaction mixtures containing 5–400 lMNBS2and 0.2 mM
NADPH in assay buffer (100 mM potassium phosphate,
pH 7.0, 2 mMEDTA), and activity was calculated from the
increase in absorbance at 412 nm using a molar extinction
coefficient of 27.2 mM )1Æcm)1, since reduction of DTNB by
1 mol of NADPH yields 2 mol of 2-nitro-5-thiobenzoate
(e412¼ 13.6 mM )1Æcm)1) In the thioredoxin reduction assay
[26], enzymes were added to the reaction mixtures containing
0.2–4 lMApTrx, 0.2 mMNADPH, and 0.5 mgÆmL)1
insu-lin in assay buffer, and activity was calculated from the
decrease in absorbance at 340nm using a molar extinction
coefficient of 6.22 mM )1Æcm)1 The Kmand Vmaxvalues were
obtained by the damped nonlinear least-squares method
(Marquardt–Levenberg method) [27,28]
Redox potential of thioredoxin
The reversibility of the reaction NADPH + TrxS2+
H+« Trx(SH)2+ NADP+was employed for
determin-ing the equilibrium constant usdetermin-ing the absorbance change at
340nm Thioredoxin (5–40lM) was mixed with 50 lM
NADPH in a total volume of 500 lL at pH 7.0 , 25C,
followed by addition of 50nM thioredoxin reductase and
then excess NADP+(1200 lM) as described previously [29]
From the equilibrium concentrations, redox potentials were
calculated according to the Nernst equation:
Eo¢(substrate) ¼ Eo¢(NADP+) + (RT/nF )
· ln([NADP+][substratered]/[NADPH][substrateox])
A value of )0.315 V was used as the redox potential of
NADP+[30]
R E S U L T S
Cloning of the two genes fromA pernix K1
In the A pernix K1 genome database (http://www.bio nite.go.jp/cgi-bin/dogan/genome_top.cgi?ape), 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 cerevisiaeand is less similar to that of E 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 140and 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-200HR 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 ApTrx 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.
Trang 4showed a visible absorption spectrum typical for
flavopro-teins with absorbance maxima at 380and 460nm (Fig 3)
The ratio between A280 and A460for the ApTR is 8.1, in
agreement with 8.0of 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 520nm as observed for E coli TR
[33,34] (Fig 3) Recombinant protein for APE0641
exhi-bited only the absorbance maximum at 280nm
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 30C and
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 coliTR (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 50m 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 380nm (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 (30kDa), 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-200HR 26/60column 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).
Trang 5ApTrx had no thiol-transferase activity by a
glutathione-disulfide oxidoreductase (data not shown)
Redox potential ofApTrx
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 50nM 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/TrxS2 redox pair has a redox
potential of)262 mV at pH 7.0and 25 C, and shows a
higher redox potential than the value reported for E coli
Trx ()270mV) [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)260to )280mV [36], and the standard state redox potential of ApTrx is contained within this range
Catalytic properties ofApTR
To obtain the kinetic parameters of ApTR for various substrates, we used the Trx and NBS2reduction assay as described in Materials and methods The kinetic parameters
of the reaction for ApTrx, NBS2, and NADPH are summarized in Table 1 The Kmof ApTR for recombinant ApTrx at pH 7.0and 25C was 12.3 ± 2.7 lM The Kmof ApTR for NADPH was 3.6 ± 0 5 lM and showed a slightly lower Km value than that for the E coli TR (4.55 lM) [37] Thus, ApTR catalyzed NADPH-dependent reductions of ApTrx and NBS2, 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 50n M
ApTR When the reaction had stopped, NADP + was added to a final concentration of 1.2 m M The formation of NADP + and NADPH was followed from the decrease and increase at 340nm, respectively.
Fig 5 Trx activity in thioredoxin systems of
A pernix and E coli The assays were
per-formed with a 50n 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 20min 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 650nm is plotted against the reaction time d,
negative control; h, 1 l M and n, 2 l M A pernix Trx; j, 1 l M and m,
2 l M E coli Trx.
Trang 6Thermophilicity and thermostability ofApTR
The thermophilicity of ApTR was investigated by
measur-ing the NBS2reductase activity at increasing temperatures
As indicated in Fig 7A, ApTR showed the maximum
activity at 90C, which is in the temperature range for
growth of A pernix [21] The thermostability of the
flavoenzyme ApTR was estimated by measuring the residual
NBS2reductase activity at 30C 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 110C 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]
D I S C U S S I O N
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
is the same as that of E 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 ()270mV) 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 ApTrx is larger than 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 Mr class NBS2 is not directly reduced by low Mr 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 Mr 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
of the enzyme were performed in parallel (B) Enzyme (1 l M ApTR in 50m 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 C in the presence (d) or absence (j) of FAD, and the residual NBS 2 reductase activity of samples were measured at 30 C.
Table 1 Kinetic parameters for ApTR 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 Data represent
the mean (±SE) of three separate experiments.
K m (l M ) k cat (S)1)
Trang 7conserved 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 450nm [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 100C (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
A C K N O W L E D G E M E N T S
S.-J Jeon was supported by the New Energy Industrial Technology
Development Organization (NEDO).
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5 Tsang, M.L & Schiff, J.A (1976) Sulfate-reducing pathway in
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