Hyperthermophilic Archaeon Aeropyrum Pernix K1 Research Articles

Doxorubicin loaded thermostable nanoarchaeosomes: a next-generation drug carrier for breast cancer therapeutics.

Breast cancer has a poor prognosis due to the toxic side effects associated with high doses of chemotherapy. Liposomal drug encapsulation has resulted in clinical success in enhancing chemotherapy tolerability. However, the formulation faces severe limitations with a lack of colloidal stability, reduced drug efficiency, and difficulties in storage conditions. Nanoarchaeosomes (NA) are a new generation of highly stable nanovesicles composed of the natural ether lipids extracted from archaea. In our study, we synthesized and characterized the NA, evaluated their colloidal stability, drug release potential, and anticancer efficacy. Transmission electron microscopy images have shown that the NA prepared from the hyperthermophilic archaeon Aeropyrum pernix K1 was in the size range of 61 ± 3 nm. The dynamic light scattering result has confirmed that the NA were stable at acidic pH (pH 4) and high temperature (70 °C). The NA exhibited excellent colloidal stability for 50 days with storage conditions at room temperature. The cell viability results have shown that the pure NA did not induce cytotoxicity in NIH 3T3 fibroblast cells and are biocompatible. Then NA were loaded with doxorubicin (NAD), and FTIR and UV-vis spectroscopy results have confirmed high drug loading efficiency of 97 ± 1% with sustained drug release for 48 h. The in vitro cytotoxicity studies in MCF-7 breast cancer cell lines showed that NAD induced cytotoxicity at less than 10 nM concentration. Fluorescence-activated cell sorting (FACS) results confirmed that NAD induced late apoptosis in nearly 92% of MCF-7 cells and necrosis in the remaining cells with cell cycle arrest at the G0/G1 phase. Our results confirmed that the NA could be a potential next-generation carrier with excellent stability, high drug loading efficiency, sustained drug release ability, and increased therapeutic efficacy, thus reducing the side effects of conventional drugs.

Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine. Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids. Method: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield. Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS. Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

Antioxidative Activity of Methanolic and Water Extracts from the Hyperthermophilic Archaeon Aeropyrum pernix K1

The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.

Role of F225 in O-phosphoserine sulfhydrylase from Aeropyrum pernix K1.

O-Phosphoserine sulfhydrylase (OPSS) synthesizes cysteine from O-phospho-L-serine (OPS) and sulfide. We have determined the three-dimensional structures of OPSS from hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) in complex with aminoacrylate intermediate (AA) formed from pyridoxal 5'-phosphate with OPS or in complex with cysteine and compared them with that of ApOPSS. We found an orientational change of F225 at the active-site entrance and constructed an F225A mutant to examine its activities and AA stability and clarify the role of F225 in ApOPSS. The OPS and O-acetyl-L-serine (OAS) sulfhydrylase activities of the F225A mutant decreased by 4.2- and 15-fold compared to those of the wild-type (wt) ApOPSS, respectively. The ability of OPS and OAS to form AA also decreased by 12- and 27-fold, respectively. AA was less stable in the F225A mutant than in the wt ApOPSS. Simulated docking showed that leaving groups, such as phosphate and acetate, were oriented to the inside of the active site in the F225A mutant, whereas they were oriented to the entrance in the wt ApOPSS. These results suggest that F225 in ApOPSS plays important roles in maintaining the hydrophobic environment of AA from solvent water and in controlling the orientation of leaving groups.

Catalytic properties and crystal structure of thermostable NAD(P)H-dependent carbonyl reductase from the hyperthermophilic archaeon Aeropyrum pernix K1.

A gene encoding NAD(P)H-dependent carbonyl reductase (CR) from the hyperthermophilic archaeon Aeropyrum pernix K1 was overexpressed in Escherichia coli. Its product was effectively purified and characterized. The expressed enzyme was the most thermostable CR found to date; the activity remained at approximately 75% of its activity after incubation for 10min up to 90°C. In addition, A. pernix CR exhibited high stability at a wider range of pH values and longer periods of storage compared with CRs previously identified from other sources. A. pernix CR catalyzed the reduction of various carbonyl compounds including ethyl 4-chloro-3-oxobutanoate and 9,10-phenanthrenequinone, similar to the CR from thyroidectomized (Tx) chicken fatty liver. However, A. pernix CR exhibited significantly higher Km values against several substrates than Tx chicken fatty liver CR. The three-dimensional structure of A. pernix CR was determined using the molecular replacement method at a resolution of 2.09Å, in the presence of NADPH. The overall fold of A. pernix CR showed moderate similarity to that of Tx chicken fatty liver CR; however, A. pernix CR had no active-site lid unlike Tx chicken fatty liver CR. Consequently, the active-site cavity in the A. pernix CR was much more solvent-accessible than that in Tx chicken fatty liver CR. This structural feature may be responsible for the enzyme's lower affinity for several substrates and NADPH. The factors contributing to the much higher thermostability of A. pernix CR were analyzed by comparing its structure with that of Tx chicken fatty liver CR. This comparison showed that extensive formation of the intrasubunit ion pair networks, and the presence of the strong intersubunit interaction, is likely responsible for A. pernix CR thermostability. Site-directed mutagenesis showed that Glu99 plays a major role in the intersubunit interaction. This is the first report regarding the characteristics and three-dimensional structure of hyperthermophilic archaeal CR.

Codon optimisation is key for pernisine expression in Escherichia coli.

BackgroundPernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.Methodology/ Principal FindingsThe challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisinewt), codon-optimised (pernisineco), and codon-optimised with a S355A mutation of a predicted active site (pernisineS355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni2+ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisinewt was not expressed in E. coli at detectable levels, while pernisineco and pernisineS355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisineco was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisineS355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.Conclusions/ SignificanceThese data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.

Structural Analysis of the Substrate Recognition Mechanism in O-Phosphoserine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1

L-Cysteine is synthesized from O-acetyl-L-serine (OAS) and sulfide by O-acetylserine sulfhydrylase (OASS; EC 2.5.1.47) in plants and bacteria. O-phosphoserine sulfhydrylase (OPSS; EC 2.5.1.65) is a novel enzyme from the hyperthermophilic aerobic archaeon Aeropyrum pernix K1 (2003). OPSS can use OAS or O-phospho-L-serine (OPS) to synthesize L-cysteine. To elucidate the mechanism of the substrate specificity of OPSS, we analyzed three-dimensional structures of the active site of the enzyme. The active-site lysine (K127) of OPSS forms an internal Schiff base with pyridoxal 5'-phosphate. Therefore, crystals of the complexes formed by the K127A mutant with the external Schiff base of pyridoxal 5'-phosphate with either OPS or OAS were prepared and examined by X-ray diffraction analysis. In contrast to that observed for OASS, no significant difference was seen in the overall structure between the free and complexed forms of OPSS. The side chains of T152, S153, and Q224 interacted with the carboxylate of the substrates, as a previous study has suggested. The side chain of R297 has been proposed to recognize the phosphate group of OPS. Surprisingly, however, the position of R297 was significantly unchanged in the complex of the OPSS K127A mutant with the external Schiff base, allowing enough space for an interaction with OPS. The positively charged environment around the entrance of the active site including S153 and R297 is important for accepting negatively charged substrates such as OPS.

Enhanced Activity and Enantioselectivity of a Hyperthermophilic Esterase from Archaeon Aeropyrum pernix K1 by Acetone Treatment

To improve the activity and enantioselectivity of hyperthermophilic archaeon Aeropyrum pernix K1 esterase (APE1547) and its mutants, they were purified by acetone-treated method. It was found that the acetone treatment not only caused APE1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by Ni-chelating column. In hydrolysis of p-nitrophenyl caprylate, the acetone-treated APE1547 and mutant A containing the following substitutions R11G, L36P, V225A, I551L, and A564T showed 5.7- and 6.9-fold active increase, respectively. In the resolution of 2-octanol acetate, the acetone-treated mutant A had a 9-fold enantioselective increase relative to that purified by Ni-chelating column. In addition, the impact of pH, temperature, and chemical reagents on activity of APE1547 and mutant A was discussed in this paper.

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (k (cat)/K (m)) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

An Efficient Method for the Expression and Reconstitution of Thermostable Mn/Fe Superoxide Dismutase from Aeropyrum pernix K1

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and over-expressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD which was active with either Fe(II) or Mn(II) as a cofactor. The ApSOD was highly stable against high temperature. This thermostable ApSOD is expected to be applicable as a useful biocatalyst for medicine and bio-industrial processes.

Outside the Unusual Cell Wall of the Hyperthermophilic Archaeon Aeropyrum pernix K1

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.

The conserved N-terminal helix of acylpeptide hydrolase from archaeon Aeropyrum pernix K1 is important for its hyperthermophilic activity

The acylpeptide hydrolases from hyperthermophilic archaeon Aeropyrum pernix K1 has a short conserved N-terminal helix in its family. The role of this N-terminal helix in the function of the hyperthermophilic enzyme, however, is unknown. Here, we investigated this question by protein engineering and biophysical methods. We found that a mutant (ΔN21) with the N-terminal helix deleted is no longer functional at the optimum temperature for WT enzyme (95 °C), required for the survival of Aeropyrum pernix K1. Instead, ΔN21 has the optimum activity at ∼ 77 °C, with higher activities than the WT enzyme below this temperature. ΔN21 is less stable than the WT enzyme and started unfolding at ∼ 77 °C, indicating that the loss of the enzymatic activity of ΔN21 at higher temperature is due to its low thermodynamic stability. In addition, we found that the salt bridges formed between the N-terminal helix and the catalytic domain of the enzyme play only a minor role in stabilizing the enzyme, suggesting that hydrophobic interactions mainly contribute to the stabilization. Since the N-terminal helix is conserved in this family of enzymes, our results suggest that the N-terminal helix is likely to play an important role for stabilizing all other enzymes in this family.

Temperature- and pH-Induced Structural Changes in the Membrane of the Hyperthermophilic Archaeon Aeropyrum pernix K1

The influence of pH and temperature on the structural organization, fluidity and permeability of the hyperthermophilic archaeon membrane was investigated in situ by a combination of electron paramagnetic resonance (EPR) and fluorescence emission spectroscopy. For EPR measurements, Aeropyrum pernix cells, after growing at different pHs, were spin-labeled with the doxyl derivative of palmitic acid methylester (MeFASL(10,3)). From the EPR spectra maximal hyperfine splitting (2A (max)) and empirical correlation time (tau (emp)), which are related to mean membrane fluidity, were determined. The mean membrane fluidity increases with temperature and depends on the pH of the growth medium. Computer simulation of the EPR spectra shows that membrane of A. pernix is heterogeneous and consists of the regions characterized with three different types of motional characteristics, which define three types of membrane domains. Order parameter and proportion of the spin probes in the three types of domains define mean membrane fluidity. The fluidity changes of the membrane with pH and temperature correlate well with the ratio between the fluorescence emission intensity of the first and third bands in the vibronic spectra of pyrene, I(1)/I(3). At pH 7.0 a decrease of I(1)/I(3) from 2.0 to 1.2, due to the penetration of pyrene into the nonpolar membrane region, is achieved at temperatures above 65 degrees C, the lower temperature limit of A. pernix growth.

Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1

This paper reports the characterization of an alkaline phosphatase (AP) from an aerobic hyperthermophilic Archaeon Aeropyrum pernix K1. The native AP was purified into homogeneity. The enzyme is predicted as a homodimeric structure with a native molecular mass of about 75 kDa and monomer of about 40 kDa. Apparent optimum pH and temperature were estimated at 10.0 and above 95 degrees C, respectively. Magnesium ion increased both the stability and the activity of the enzyme. A. pernix AP has been demonstrated as a very thermostable AP, retaining about 76% of its activity after being incubated at 90 degrees C for 5.5 h and 67% of its activity after being incubated at 100 degrees C for 2.5 h, respectively, under the presence of Mg(II). Enzyme activity was increased in addition of exogenous Mg(II), Ca(II), Zn(II), and Co(II).

Overexpression, purification and crystallization of tyrosyl-tRNA synthetase from the hyperthermophilic archaeon Aeropyrum pernix K1.

Hyperthermophilic archaeal tyrosyl-tRNA synthetase from Aeropyrum pernix K1 was cloned and overexpressed in Escherichia coli. The expressed protein was purified by Cibacron Blue affinity chromatography following heat treatment at 363 K. Crystals suitable for X-ray diffraction studies were obtained under optimized crystallization conditions in the presence of 1.5 M ammonium sulfate using the hanging-drop vapour-diffusion method. The crystals belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 66.1, c = 196.2 A, and diffracted to beyond 2.15 A resolution at 100 K.

Crystallization and preliminary X-ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

Thioredoxin peroxidase is a member of the peroxiredoxin family and plays a dominant role in a hydrogen peroxide metabolism. A recombinant form of the hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1, a polypeptide consisting of 250 amino acids, was purified. The C207S mutant protein was crystallized by the hanging-drop vapour-diffusion method using potassium sodium tartrate as the precipitant at 298 K. Diffraction data were collected and processed to 2.7 A resolution. The crystal belongs to space group P1, with unit-cell parameters a = 126.2, b = 126.3, c = 213.7 A, alpha = 80.4, beta = 80.3, gamma = 70.7 degrees. Calculation of the self-rotation function showed that the protein quaternary structure includes a fivefold axis and five twofold axes.

Properties of an Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Aeropyrum pernix K1

A NAD+-dependent medium-chain alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix K1 was expressed in Escherichia coli and purified. The recombinant enzyme was a homotetramer of molecular mass 1.6×102 kDa. The optimum pH for the oxidative reaction was around 10.5 and that for the reductive reaction was around 8.0. The enzyme had a broad substrate specificity including aliphatic and aromatic alcohols, aliphatic and aromatic ketones, and benzylaldehyde. This enzyme produced (S)-alcohols from the corresponding ketones. The enzyme was thermophilic and the catalytic activity increased up to 95°C. It maintained 24% of the original catalytic activity after incubation for 30 min at 98°C, indicating that this enzyme is highly thermostable.

A novel ADP-dependent DNA ligase from Aeropyrum pernix K1

A gene encoding a putative ATP-dependent DNA ligase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (accession no. APE1094) from A. pernix encoding a 69-kDa protein showed a 39–61% identity with other ATP-dependent DNA ligases from the archaea. Normally DNA ligase is activated by NAD + or ATP. There has been no report about the other activators for DNA ligase. The recombinant ligase was a monomeric protein and catalyzed strand joining on a singly nicked DNA substrate in the presence of ADP and a divalent cation (Mg 2+, Mn 2+, Ca 2+ and Co 2+) at high temperature. The optimum temperature and pH for nick-closing activity were above 70°C and 7.5°C, respectively. The ligase remained stable for 60 min of treatment at 100°C, and the half-life was about 25 min at 110°C. This is the first report of a novel hyperthermostable DNA ligase that can utilize ADP to activate the enzyme.

Characterization of a novel thermostable O-acetylserine sulfhydrylase from Aeropyrum pernix K1.

An O-acetylserine sulfhydrylase (OASS) from the hyperthermophilic archaeon Aeropyrum pernix K1, which shares the pyridoxal 5'-phosphate binding motif with both OASS and cystathionine beta-synthase (CBS), was cloned and expressed by using Escherichia coli Rosetta(DE3). The purified protein was a dimer and contained pyridoxal 5'-phosphate. It was shown to be an enzyme with CBS activity as well as OASS activity in vitro. The enzyme retained 90% of its activity after a 6-h incubation at 100 degrees C. In the O-acetyl-L-serine sulfhydrylation reaction, it had a pH optimum of 6.7, apparent K(m) values for O-acetyl-L-serine and sulfide of 28 and below 0.2 mM, respectively, and a rate constant of 202 s(-1). In the L-cystathionine synthetic reaction, it showed a broad pH optimum in the range of 8.1 to 8.8, apparent K(m) values for L-serine and L-homocysteine of 8 and 0.51 mM, respectively, and a rate constant of 0.7 s(-1). A. pernix OASS has a high activity in the L-cysteine desulfurization reaction, which produces sulfide and S-(2,3-hydroxy-4-thiobutyl)-L-cysteine from L-cysteine and dithiothreitol.

Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1.

We have identified and characterized a thermostable thioredoxin 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 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 thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 degrees C, and the half-life of heat inactivation was about 4 min at 110 degrees 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 proteins. 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. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.