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: 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) 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 (http://www.bio. 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. 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Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1 Sung-Jong Jeon and Kazuhiko Ishikawa National Institute of Advanced. thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria. Keywords: thioredoxin; thioredoxin reductase; hyper- thermophile;