INTRODUCTION

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During the past decade, many anaerobic hyperthermophiles growing at a temperature near or above the boiling point of water have been isolated from marine and continental volcanic environments (1). The interest in hyperthermophiles has been rapidly expanding. In particular, interest is focused on understanding the adaptation mechanisms that allow the metabolism to function and the biomolecules, such as protein, enzyme, and DNA, to remain intact at extremely high temperature. Most hyperthermophiles belong to Archaea, the third domain of life (22), and evolutionary attention has been paid to their biomolecules because they may be the most slowly evolving or primitive group of microorganisms yet discovered. In addition, enzymes from the hyperthermophiles have a large biotechnological potential (2, 6). Of the enzymes from hyperthermophiles, glutamate dehydrogenase (GluDH) (EC 1.4.1.4., glutamate:NADP oxidoreductase) is one of the enzymes for which the most abundant information concerning enzymological properties and the relationships between structure and function has been obtained. Extremely thermostable NADP-dependent GluDHs have been purified from Pyrococcus furiosus (5, 18, 20), Pyrococcus woesei (18), Thermococcus litoralis (14, 19), and Thermococcus profundus (11). The gdhA gene of Pyrococcus furiosus (8, 9) has been cloned and sequenced, and the structural difference between the GluDHs of Pyrococcus furiosus, T. litoralis, and Clostridium symbiosum has been investigated to elucidate protein thermostability (3). In addition, a key role of the ion pair networks in maintaining the structure stability of Pyrococcus furiosus GluDH at an extremely high temperature has been indicated (24). However, information about hyperthermostable GluDH is limited so far to that regarding marine hyperthermophilic species of the order Thermococcales such as Pyrococcus and Thermococcus.

In the course of investigating GluDH distribution in hyperthermophilic archaea, we found the activity of NAD-dependent GluDH (EC 1.4.1.2) in the cell extract of a continental hyperthermophilic archaeon, Pyrobaculum islandicum. This is the first example of the occurrence of NAD-dependent GluDH in anaerobic hyperthermophilic archaea. In general, the physiological function of NAD-dependent GluDH is known to be different from that of NADP-dependent GluDH (17). In addition, the NAD-dependent GluDH may be expected to be more preferable for application than the NADP-dependent enzyme, because NAD and NADH are much cheaper than NADP and NADPH, respectively (4, 23). Thus, we purified the enzyme from P. islandicum for characterization. We describe here the characteristics of this GluDH with emphasis on its high stability in some denaturants and organic solvents.

	MATERIALS AND METHODS

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Chemicals and biochemicals. NAD, NADH, NADP, and NADPH were obtained from the Kojin Co., Tokyo, Japan. All analytical-grade reagents, such as L-glutamate monosodium salt, sodium 2-oxoglutarate, urea, guanidine hydrochloride, and acetonitrile, were purchased from Nacalai Tesque, Kyoto, Japan, or Wako Pure Chemicals, Osaka, Japan. Red Sepharose CL-4B (reactive red 120 dye; Sigma) was prepared as previously described (16).

Microorganism and growth conditions. The hyperthermophilic archaeon P. islandicum DSM 4184 was obtained from the German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Göttingen, Germany. P. islandicum was cultured in a medium containing 6.5 g of (NH₄)₂SO₄, 0.28 g of KH₂PO₄, 0.25 g of MgSO₄ · 7H₂O, 0.07 g of CaCl₂ · 2H₂O, 0.02 g of FeCl₃ · 6H₂O, 1.8 mg of MnCl₂ · 4H₂O, 4.5 mg of Na₂B₄O₇ · 10H₂O, 0.22 mg of ZnSO₄ · 7H₂O, 0.05 mg of CuCl₂ · 2H₂O, 0.03 mg of Na₂MoO₄ · 2H₂O, 0.03 mg of VOSO₄ · 2H₂O, 0.01 mg of CoSO₄, 2 g of Na₂S₂O₃ · 5H₂O,1 mg of resazurin, 2.5 g of polypeptone, 1 g of yeast extract, and 0.5 g of Na₂S · 9H₂O per liter (pH 6.0 adjusted with 10 N H₂SO₄). Dissolved oxygen was removed from the medium by an aspirator, and then liquid paraffin was layered on the surface of the medium to prevent its contact with air. Anaerobic conditions were achieved by flushing the medium with N₂ gas. The seed culture, about 10% volume of the medium, was inoculated into a bottle filled with the medium, and the bottle was incubated at 90°C for about 4 days on a hot plate with stirring with a magnetic bar. The cells were collected by centrifugation (10,000 × g for 10 min) and washed twice with 0.85% NaCl solution. The washed cells were suspended in 10 mM potassium phosphate buffer (pH 7.2) and stored at 20°C.

Enzyme assay and protein determination. Enzyme activity was assayed spectrophotometrically with a Shimadzu 160A spectrophotometer equipped with a thermostat. The standard reaction mixture for oxidative deamination was composed of 200 µmol of glycine-KOH buffer (pH 9.7), 10 µmol of L-glutamate (pH 9.7), 1.25 µmol of NAD, and the enzyme in a final volume of 1.00 ml. For reductive amination, the mixture contained 200 µmol of glycine-KOH buffer (pH 8.7), 200 µmol of NH₄Cl (pH 8.7, adjusted with KOH), 10 µmol of sodium 2-oxoglutarate, 0.20 µmol of NADH, and the enzyme in a total volume of 1.00 ml. After the reaction mixture without the coenzyme was incubated at 50°C for 5 min, the reaction was started by the addition of the coenzyme. The increase and decrease of NADH were monitored by absorbance at 340 nm. One unit of the enzyme is defined as the amount catalyzing the formation of 1 µmol of NADH per min at 50°C in the oxidative deamination of L-glutamate. The protein concentration was determined by the spectrophotometric method of Kalb and Bernlohr (10). With the column fractions of Red Sepharose CL-4B chromatography, the protein was monitored by absorbance at 280 nm.

Purification of glutamate dehydrogenase from P. islandicum. The entire operation was performed at room temperature (about 25°C). Glycerol (10%) was added to all buffers used in purification steps. The cells were disrupted by ultrasonication, the cell debris was removed by centrifugation (20,000 × g, 10 min), and the supernatant solution was used as the crude extract for the purification.

PAGE. Polyacrylamide gel electrophoresis (PAGE; 7.5% acrylamide gel) was performed by the method of Davis (7), and sodium dodecyl sulfate (SDS)-PAGE (12% acrylamide slab gel, 1-mm thick) was carried out by the procedure of Laemmli (12). Activity staining was carried out at 50°C in a mixture containing 0.2 M Tris-HCl buffer (pH 8.0), 10 mM L-glutamate, 1.0 mM NAD, 0.04 mM phenazine methosulfate, and 0.05 mM p-iodonitrotetrazolium violet until a red band of sufficient intensity was visible. The protein band was stained with Coomassie brilliant blue G-250 (PAGE) and R-250 (SDS-PAGE).

Molecular mass determinations. The molecular mass of the native enzyme was determined by high-performance liquid chromatography (HPLC; Tosoh type CCPE) with a gel filtration column (TSKgel column G3000SW_XL; 7.8 mm by 30 cm). The column was equilibrated with 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 M Na₂SO₄ and 0.05% NaN₃. The following standard proteins (Bio-Rad) were used to make a calibration curve: bovine thyroglobulin (molecular mass, 670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa), and vitamin B₁₂ (1,350 Da). SDS-PAGE was used for the molecular mass determination of the subunit. The marker proteins (New England Biolabs) used were as follows: fusion protein of maltose-binding protein and -galactosidase (molecular mass, 175 kDa), fusion protein of maltose-binding protein and paramyosin (83 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa), triosephosphate isomerase (32.5 kDa), -lactoglobulin A (25 kDa), and lysozyme (16.5 kDa).

Amino acid sequencing. Three nanomoles of the purified GluDH was digested with 3 nmol of lysyl endopeptidase (Wako Pure Chemicals) at 30°C overnight. The digested peptides were purified by HPLC with a reverse-phase column (Symmetry C₁₈; Waters). The peptides were eluted with a linear gradient of acetonitrile (5 to 60%) containing 0.05% trifluoroacetic acid. Elution of the peptides was monitored by absorbance at 220 nm. The amino acid sequence of the peptide was determined by automated Edman degradation using an Applied Biosystems 473A protein sequencer.

Steady-state kinetic analyses. The basic reaction mixtures were similar to those described in "Enzyme assay and protein determination." Initial velocity experiments were done by varying the concentration of one substrate while keeping the concentrations of the other substrates fixed as previously described (15). The K_m values were calculated from the secondary plot of the intercepts versus the reciprocal of the substrate concentration.

Activity and stability of the enzyme in denaturants, water-miscible organic solvents, and detergents. The effects of detergents, denaturants, or organic solvents on the enzyme activity were detected by measuring the activity in the presence of these reagents in the standard assay system (glutamate deamination). In the case of organic solvents, the reaction mixture in the spectrophotometer cuvette was shielded with a Teflon cap and Parafilm. The effects of detergents, denaturants, or organic solvents on enzyme stability were examined by measuring the activity remaining by incubation with these reagents. An aliquot of the incubation mixture was withdrawn, and the remaining activity of the enzyme was assayed at 50°C. The denaturants used were guanidine hydrochloride and urea. The water-miscible organic solvents used were acetonitrile, methanol, ethanol, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and N,N-dimethyl formamide (DMF). The detergents used were Triton X-100 and sodium deoxycholic acid (DOC).

	RESULTS

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Purification. Table 1 shows a typical result of purification of GluDH from the extract of P. islandicum. The enzyme was purified about 250-fold with a 61% recovery by subjecting it to two successive Red Sepharose CL-4B affinity chromatographies within a few days. In the first column chromatography, the enzyme was released from the affinity resin by the nonspecific elution method by increasing the NaCl concentration. This method was useful for the rapid removal of a large amount of contaminant protein. In the second column chromatography, specific affinity elution by the ternary complex formation of NAD-enzyme-L-glutamate was used and achieved very high resolution. The purified enzyme was found to be homogeneous on the basis of SDS-PAGE (Fig. 1).

View larger version (34K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE of purified P. islandicum GluDH. Lanes: 1, molecular mass standards; 2, GluDH.

Molecular mass and subunit structure. The molecular mass of the P. islandicum GluDH was determined to be about 220 kDa by gel filtration. SDS-PAGE of the purified enzyme gave only one band; the subunit molecular mass was estimated to be about 36 kDa (Fig. 1). Thus, the native enzyme probably has a hexamer structure composed of six identical or similar subunits.

Amino acid sequence. The N-terminal sequence of the purified enzyme was analyzed several times by automated Edman degradation but could not be detected. The amino acid sequence of an inner peptide (40 amino acids) was determined and aligned with those of the enzymes from other origins (Fig. 2). Computer comparison of the sequence of P. islandicum enzymes with those of other GluDHs in the SwissProt database revealed that the enzyme sequence exhibited the highest similarity with those of Sulfolobus shibatae and Sulfolobus solfataricus enzymes (52.5%). In addition, high sequence similarity was observed between this enzyme and enzymes from the following organisms: Pyrococcus furiosus, Thermococcus strain ES4, and Clostridium difficile (45%); Halobacterium salinarium (42.5%); and chicken, cow, mouse, and human (40%).

View larger version (61K): [in this window] [in a new window]   FIG. 2.   Internal amino acid sequence of GluDH from P. islandicum and comparison with other GluDH sequences. P. is, P. islandicum; S. shi, S. shibatae; S. sol, S. solfataricus; P. fur, Pyrococcus furiosus; T. ES4; Thermococcus strain ES4, T. lit, T. litoralis; P. asa, Peptostreptococcus asaccharolyticus, C. dif, C. difficile; H. sal; H. salinarium.

Effects of temperature and pH on the enzyme activity. The effect of temperatures in the range of 30 to 100°C on oxidative deamination was investigated. The activity of the enzyme increased with an increase in temperature from 30 to 90°C. The activity observed at 30°C was less than several percent of that at 50°C. The highest activity was observed around 90°C and was about seven times higher than that at 50°C. The activity at 100°C was about 80% of the highest activity at 90°C.

Stability. The thermostability of the enzyme was examined. The enzyme retained its full activity after heating at temperatures from 50 to 100°C for 10 min but completely lost activity after incubation at 110°C for 10 min. The addition of 0.5 M NaCl or 0.5 M KCl to the enzyme solution did not affect the thermostability. With heat treatment at 100°C, the enzyme activity was not lost for at least 2 h.

Substrate and coenzyme specificity and kinetic constants. The ability of the enzyme to catalyze the oxidative deamination of various -amino acids and the reductive amination of various 2-oxo acids was examined (Table 2). The enzyme reacted mainly with L-glutamate in oxidative deamination. In addition, the enzyme catalyzed the oxidative deamination of several -amino acids such as L-norvaline, L-2-aminobutyrate, and L-valine with a low reaction rate. For reductive amination, 2-oxoglutarate was the most preferred substrate. The enzyme catalyzes the reductive amination of several 2-oxo acids, such as 2-oxovalerate, 2-oxoisocaproate, and 2-oxobutyrate.

Effects of denaturants, water-miscible organic solvents, and detergents on activity and stability. The effects of guanidine hydrochloride and urea on the enzyme activity were examined with an assay at 50°C. Enzyme activity was remarkably enhanced by the addition of guanidine hydrochloride and urea (Fig. 3A). The addition of 0.8 M guanidine hydrochloride gave the maximum enhancement (about 370%). The activity was almost lost by the addition of more than 6 M guanidine hydrochloride. In the case of urea addition, the maximum activity was observed with a concentration of 5 to 6 M but remarkable enhancement, as in the case of guanidine hydrochloride, was not observed.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   (A) Effects of denaturants on GluDH activity. The effect of the enzyme on oxidative deamination was assayed at 50°C. The reaction was started by the addition of the enzyme to the reaction mixture containing various concentrations of urea () or guanidine hydrochloride (). (B) Effects of denaturants on GluDH stability. The enzyme was incubated with various concentrations of urea at 50°C () and 90°C () for 10 min and of guanidine hydrochloride at 50°C () and 90°C () for 10 min. After incubation, the activity of the aliquot on oxidative deamination was assayed at 50°C.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Change in fluorescence intensity at 335 nm of P. islandicum GluDH by incubation with denaturant. The enzyme was incubated with various concentrations of urea at 50°C () and 90°C () for 15 min and of guanidine hydrochloride at 50°C () and 90°C () for 15 min. Fluorescence emission was monitored at 50°C. The excitation wavelength was 280 nm.

View larger version (23K): [in this window] [in a new window]   FIG. 5.   (A) Effects of water-miscible organic solvents on P. islandicum GluDH activity. The enzyme activity on oxidative deamination was assayed at 50°C. The reaction was started by the addition of the enzyme to the reaction mixture containing various concentrations of organic solvent. (B) Effects of water-miscible organic solvents on P. islandicum GluDH stability. The enzyme was incubated with various concentrations of organic solvents at 50°C for 10 min. After incubation, the activity of the aliquot on oxidative deamination at 50°C was assayed. The organic solvents used were ethanol (), methanol (), acetonitrile (), THF (), DMF (), and DMSO ().

View larger version (20K): [in this window] [in a new window]   FIG. 6.   Effects of detergents on P. islandicum GluDH stability. The enzyme was incubated with various concentrations of Triton X-100 at 50°C () and 90°C () for 10 min and of DOC at 50°C () and 90°C () for 10 min. The activity of the aliquot on oxidative deamination at 50°C was assayed.

	DISCUSSION

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In this study, GluDH from the continental hyperthermophilic archaeon P. islandicum has been purified and characterized. This is the first report on the purification and characterization of a continental anaerobic hyperthermophile GluDH. All of the hyperthermophile GluDHs purified and characterized to date are from the marine anaerobic hyperthermophilic species of the order Thermococcales, such as Pyrococcus furiosus (5, 18, 20), Pyrococcus woesei (18), and T. litoralis (14, 19). GluDHs from the Thermococcales utilize exclusively NADP (EC 1.4.1.4) as a coenzyme, and their principal function is suggested to be L-glutamate biosynthesis coupled with L-alanine production (11, 18). In contrast, we indicate here that the P. islandicum GluDH apparently requires NAD as a coenzyme. Based on this, the P. islandicum GluDH is distinct from those from other hyperthermophilic archaea. Seling and Schönheit (21) have suggested the presence of the citric acid cycle and its function for the oxidation of organic compounds to CO₂ with elemental sulfur or thiosulfate as the electron acceptor in P. islandicum. Thus, it is predicted that the GluDH in the cell of P. islandicum, belonging to the order Thermoproteales, links to the citric acid cycle via 2-oxoglutarate, although the physiological function of the GluDH is not yet clear. The presence of the citric acid cycle in cells of members of the Thermococcales such as Pyrococcus and Thermococcus has not yet been reported, and GluDH is abundant in the cytoplasm of cells of species of the Thermococcales, reaching more than several percent of total proteins (5, 9, 18). In contrast, the P. islandicum GluDH is not abundant (about 0.4%), as shown in Table 1. This suggests that the physiological role of the GluDH in P. islandicum is different from that of the other species of Thermococcales.

The P. islandicum GluDH consists of six subunits with identical molecular masses, and the subunit structure is similar to those of other species of the Thermococcales. However, the molecular masses of the P. islandicum enzyme (220 kDa) and its subunits (36 kDa) are slightly smaller than those of Pyrococcus furiosus, Pyrococcus woesei, T. litoralis, and T. profundus enzymes (native, 263 to 300 kDa; subunits, 38 to 43 kDa) (5, 11, 14, 18-20). This suggests diversity in the molecular structure of the GluDHs from hyperthermophiles. We could not detect the N-terminal sequence of the P. islandicum GluDH. The N-terminal sequence of the GluDH may be blocked in a different way than the P. furiosus (18) and T. litoralis GluDHs are (19), which suggests another area of diversity. On the other hand, we have determined the amino acid sequence of an inner peptide of the P. islandicum GluDH and the peptide was identified as a downstream region of a coenzyme binding site of the GluDH (24). From the sequence comparison, we have found that the P. islandicum GluDH has high sequence similarity with the enzymes from species belonging to the Archaea such as S. shibatae and Pyrococcus furiosus and from vertebrates but not from many species of bacteria except for C. difficile.

As might be expected, the P. islandicum GluDH is extremely thermostable. The enzyme is not inactivated by incubation at 100°C for 2 h. It is slightly less thermostable than the NADP-dependent GluDHs of Pyrococcus furiosus and Pyrococcus woesei (18) but more thermostable than the T. litoralis (19) and T. profundus (11) enzymes. The P. islandicum GluDH is probably the most thermostable NAD-dependent dehydrogenase among the NAD-dependent dehydrogenases from many other organisms described to date. The most thermostable NAD-dependent dehydrogenase so far reported is L-malate dehydrogenase from Archaeoglobus fulgidus. This enzyme is stable up to 90°C but loses activity at 100°C (13). The P. islandicum enzyme is also highly resistant to denaturants, organic solvents, and detergents, such as guanidine hydrochloride, urea, ethanol, methanol, DMF, and DOC, at 50°C. This suggests that the NAD-dependent GluDH of P. islandicum may be preferred in applications as a reagent for biosensor and bioreactor processes under some special conditions.

The optimum temperature for the oxidative deamination of P. islandicum GluDH is around 90°C and is similar to those of the T. litoralis and T. profundus enzymes (11, 14, 19). The optimum pHs for the oxidative deamination (9.7) and the reductive amination (8.7) of P. islandicum GluDH are more alkaline than those of the Pyrococcus furiosus, Pyrococcus woesei, and T. litoralis enzymes (5, 11, 14, 18-20). The enzyme catalyzes L-norvaline, L--aminobutyrate, and L-valine, as well as L-glutamate, in oxidative deamination, and therefore the substrate specificity is slightly low in comparison to those of NADP-dependent enzymes of other hyperthermophiles which specifically catalyze L-glutamate. In addition, low K_m values for L-glutamate, 2-oxoglutarate, and ammonia in the P. islandicum enzyme are recognized. Another remarkable characteristic of the enzyme is the enhancement of the activity with guanidine hydrochloride, urea, acetonitrile, and THF. The enzyme activity is enhanced about two to four times with 0.8 M guanidine hydrochloride, 6 M urea, 15% acetonitrile, and 10% THF. Such enhancement has not been reported for GluDHs from other hyperthermophiles, although activity enhancement with salts, such as NaCl and KCl, has been described (14, 18). In contrast, the activity of P. islandicum GluDH is not enhanced by the addition of such salts. The fluorescence emission spectrum of the enzyme without denaturants exhibits a maximum at 335 nm. When the enzyme is incubated with urea, a change in the emission spectrum is not observed. On the other hand, a significant spectrum change is observed when the enzyme is incubated with more than 4 M guanidine hydrochloride at 50°C or with more than 3 M guanidine hydrochloride at 90°C. These results suggest that the P. islandicum GluDH may have networks of ion pairs and that these networks have an important role in its hyperthermostability, like they do in Pyrococcus furiosus GluDH (24).

These characteristics of the hyperthermostable NAD-dependent GluDH from P. islandicum will assist us in elucidating the molecular basis of the mechanisms of the extremely high thermal stability and the catalytic reaction. Recently, many analyses of thermal denaturation and activation, gene cloning, and three-dimensional structure have been carried out for the NADP-dependent enzymes of Pyrococcus furiosus (3, 8, 9). In addition, these results indicate that the hyperthermostable NAD-dependent GluDH may have a high potential application to a novel bioprocess (17). The gene cloning and structure analyses of the enzyme are under investigation. The results will provide us with a further understanding of the relationship between the function and structure of the hyperthermostable enzyme and afford us further potential applications.