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NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties
FEMS Microbiology Letters 159 (1998) 15^20
NAD-speci¢c glutamate dehydrogenase from Thermus thermophilus HB8: puri¢cation and enzymatic properties Joseè Luis Ruiz, Juan Ferrer, Moènica Camacho, Mar|èa Joseè Bonete * Divisioèn de Bioqu|èmica, Facultad de Ciencias, Universidad de Alicante, Ap. 99, 03080 Alicante, Spain Received 27 August 1997; revised 25 November 1997; accepted 26 November 1997
Abstract An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85^90³C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented Km values for NAD and L-glutamate of 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72³C. Differential scanning calorimetry revealed a tm of 95³C for denaturation. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : NAD-glutamate dehydrogenase ; Thermus thermophilus; Thermostability
1. Introduction Glutamate dehydrogenases (L-glutamate NAD(P) oxidoreductase; GDHs, EC 1.4.1.2^4) have been isolated from a variety of species. The enzymes catalyse the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia using NAD or NADP as cofactors [1] according to the following reaction: l3Glutamate NAD P H2 OH 23oxoglutarate NH 4 NAD PH H
Glutamate dehydrogenases are important enzymes in * Corresponding author. Tel.: +34 (6) 590 3524; Fax: +34 (6) 590 3464; E-mail: [email protected]
the metabolism of most organisms since they provide a link between carbon and nitrogen metabolism [1]. They are involved in ammonia assimilation and catabolism of amino acids in a wide range of organisms. Three di¡erent glutamate dehydrogenases have been described: an NAD-speci¢c enzyme (EC 1.4.1.2), an NADP-speci¢c enzyme (EC 1.4.1.4) and an NAD(P)-dependent enzyme (EC 1.4.1.3). In general, prokaryotic glutamate dehydrogenase enzymes are speci¢c for either NAD in the case of catabolic enzymes or NADP in the case of biosynthetic GDHs. Enzymes puri¢ed from vertebrates can use both coenzymes. Fungi and some halophiles have two distinct glutamate dehydrogenases with kinetic properties that allow them to have only one physiological role [2^4]. Glutamate dehydrogenase is evolutionarily conserved in all three primary domains:
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Eucarya, Bacteria and Archaea, and information is available on structural, enzymatic and phylogenetic properties. We examined the activity of glutamate dehydrogenase in the cell homogenate of Thermus thermophilus, a Gram-negative aerobic microorganism, which can grow at temperatures above 75³C, and is one of the most widely studied thermophilic Bacteria [5]. This extreme thermophile belongs to one of the oldest phylogenetic branches of the Bacteria domain [6]. Cellular components of members of the genus Thermus are generally more resistant to heat and most common protein denaturants than their counterparts among mesophilic bacteria, which makes them extremely interesting for industrial processes. The genus Thermus is receiving considerable attention for its biotechnological potential [7]. Therefore, GDH from this microorganism is an attractive choice to study evolutionary aspects of enzyme structure, function and stability. In this report, we describe the puri¢cation and biochemical characterisation of the NAD-dependent glutamate dehydrogenase from T. thermophilus.
2. Materials and methods 2.1. Organisms T. thermophilus HB8 cells were kindly supplied by Dr. Joseè Berenguer from Centro Nacional de Biotecnolog|èa `Severo Ochoa' (Madrid). 2.2. Enzyme puri¢cation Frozen cells of T. thermophilus (30 g) were suspended in 150 ml of 200 mM Tris-HCl bu¡er (pH 8) containing 2 mM EDTA and 2 mM 2-mercaptoethanol to preserve GDH activity during storage and 1 mM phenylmethylsulfonyl £uoride (PMSF), a serine protease inhibitor, to prevent proteolysis. Cells were disrupted by mechanical and sonic treatment. Cell debris were removed by centrifugation at 12 000Ug at 4³C for 30 min. The supernatant was centrifuged at 105 000Ug for 2 h. The new supernatant was used for enzyme puri¢cation. With the exception of (NH4 )2 SO4 precipitation, all puri¢cation steps were carried out at room temperature.
Step 1: Solid (NH4 )2 SO4 was added to crude extract with mild stirring at 4³C up to 35% saturation. Stirring was continued for 45 min and the precipitate was collected and dissolved in 50 mM Tris-HCl buffer (pH 8), containing 2 mM EDTA and 2 mM 2mercaptoethanol (TM bu¡er). Step 2: After desalting through dialysis against TM bu¡er, the enzyme solution was loaded onto an anion-exchange column (5U2.5 cm) of Q-Sepharose (Pharmacia) equilibrated with TM bu¡er. The enzyme was eluted with a 200-ml linear gradient of 0^1 M NaCl in TM bu¡er. Fractions containing enzyme activity were pooled and dialysed against TM bu¡er. Step 3: The clear solution was applied onto a Blue-Sepharose CL-6B column (7U2.5 cm), equilibrated and washed with TM bu¡er. The enzyme was eluted with the same bu¡er containing 3 mM NAD. The active fractions were pooled. Step 4: The solution of the previous step was applied onto the Q-Sepharose column as in step 2. Step 5: Finally, the high-speci¢c-activity material from step 4 was applied onto a column (64U2.5 cm) of G-100 Sephadex (Pharmacia) equilibrated with TM bu¡er (pH 8). The enzyme was eluted from the column with the same bu¡er. The most active fractions were pooled. 2.3. Assay of glutamate dehydrogenase and protein determinations The activity of GDH was assayed spectrophotometrically at 340 nm and 72³C in 50 mM Tris-HCl bu¡er (pH 8), 2 mM EDTA containing 100 mM Lglutamate and 3 mM NAD. After equilibration at 72³C for 5 min, the reaction was started by the addition of NAD. One unit of enzyme activity is the reduction of 1 Wmol of NAD per minute. The values of Vmax and Km were determined using Cleland programs [8]. Protein concentrations were determined applying the method of Bradford. 2.4. Determination of native Mr values The Mr for the native glutamate dehydrogenase was estimated by gel ¢ltration on a Sepharose CL6B (40U2.5 cm) column. The ¢ltration was carried out using a 50 mM Tris-HCl bu¡er (pH 8) contain-
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ing 2 mM EDTA and 2 mM 2-mercaptoethanol for the standard protein markers and the Thermus glutamate dehydrogenase. Protein markers were: bovine glutamate dehydrogenase (330 kDa), catalase (240 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa) and cytochrome c (12.5 kDa). 2.5. Electrophoresis The subunit Mr of the puri¢ed glutamate dehydrogenase was determined by SDS-PAGE. Native PAGE was carried out at pH 8.9 on a 7.5% separation gel. Glutamate dehydrogenase was visualised by coupling the glutamate-dependent reduction of NAD with diaformazan production. After electrophoresis, gels were incubated in the dark for 10 min at 72³C in a solution of 50 mM Tris-HCl (pH 8) containing 100 mM L-glutamate, 3 mM NAD, 0.05% (w/v) nitroblue tetrazolium (Sigma) and 0.005% (w/v) phenazine methosulfate (Sigma), followed by ¢xing in ethanol, acetic acid, glycerol and water (5:2:1:4) for storage. 2.6. Di¡erential scanning calorimetry Scanning calorimetric measurements were carried out on a Setaram DSC92 scanning microcalorimeter at a heating rate of 1³K min31 . All measurements were performed under a helium atmosphere at a total pressure of 3 bar. Protein samples were extensively dialysed against 50 mM Tris-HCl bu¡er pH 8.0. The protein concentration in these experiments was 1.4 mg ml31 . The bu¡er-bu¡er baseline was obtained at the same scanning rate and was subtracted from the sample curve.
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3. Results The result of a typical puri¢cation procedure is summarised in Table 1. Cell-free extracts of T. thermophilus contained signi¢cant amounts of GDH activity. The extract was very stable at 8³C and 23³C; no activity was lost even after 36 days if the extract was maintained in 200 mM Tris-HCl bu¡er (pH 8) containing 2 mM EDTA, 2 mM 2-mercaptoethanol and 1 mM PMSF. The elution of the NAD-GDH from the anion-exchange column, followed by direct application onto the Blue-Sepharose CL-6B column, was crucial for the puri¢cation procedure. Triazine dyes have proved useful for the isolation of NADdependent enzymes [9]. The ¢nal preparation had a speci¢c activity of 70 U mg31 and the enzyme was puri¢ed 350-fold. The thermophilic enzyme was found to be homogeneous as judged by electrophoresis on SDS-PAGE. We also carried out analytical polyacrylamide gel electrophoresis. The enzyme migrated as a single component, and only one band of GDH activity was found through activity staining. The enzymatic activity corresponded to the stained protein band obtained with Coomassie staining. Electrophoresis of the puri¢ed GDH from T. thermophilus with anionic (SDS) detergent revealed that the enzyme migrated as a double protein band (Fig. 1). The mobility of the enzyme in SDS-PAGE corresponded to subunits Mr of 50 þ 3 kDa and 48 þ 3 kDa. An apparent molecular mass of 289 þ 30 kDa was found for the puri¢ed GDH, as estimated by gel ¢ltration, so we can conclude that glutamate dehydrogenase from T. thermophilus consists of six subunits. The enzyme speci¢cally required NAD and L-glutamate as the coenzyme and substrate respectively for the deaminating activity. T. thermophilus GDH is catalytically active only with NAD as a coenzyme
Table 1 Puri¢cation of NAD-glutamate dehydrogenase from T. thermophilus Step
Enzyme activity (U)
Total protein (mg)
Speci¢c activity (U mg31 )
Puri¢cation (n-fold)
Yield (%)
Crude extract Ammonium sulfate 35% Q-Sepharose Blue-Sepharose Q-Sepharose Sephadex G-100
250 145 110 36 30 14
1473 168 32 1 0.54 0.20
0.2 0.9 3.4 36 56 70
1 5 17 180 275 350
100 59 44 14 12 6
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Fig. 1. SDS-PAGE of T. thermophilus glutamate dehydrogenase. Lane 1, 5 Wg of protein markers (middle range of Promega) ; lane 2, 3 Wg of pooled fractions after Sephadex G-100.
and it could not be replaced by NADP. Kinetic parameters for the deamination oxidative reaction were determined under conditions of 72³C and pH 8 (in TM bu¡er). The Km values for NAD and L-glutamate were 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. The amino acids L-leucine, L-valine and L-isoleucine were tested as substrates, but were not oxidised by GDH. On the other hand, no activity in the direction of L-glutamate biosynthesis (amination of 2-oxoglutarate) was detected. The dependence on pH of the puri¢ed GDH activity was examined at 72³C using di¡erent bu¡ers ranging from pH 5 to pH 10. All pH values were corrected as regards the temperature e¡ect on pH. Optimal activity occurred in 50 mM Tris-HCl bu¡er pH 8.0, containing 2 mM EDTA. The temperature dependence of Vmax (deamination reaction) was determined over 35^95³C and documented as an Arrhenius plot (Fig. 2). Optimal activity was observed at 85^90³C. Taking into account the temperature activity dependence, the activation energy (Ea ) for the oxidative deamination of L-glutamate was found to be 36 kJ mol31 . Thermal inactivation kinetics were measured at di¡erent temperatures. Heat-induced inactivation kinetics of the enzyme ¢t very well into an irreversible ¢rst-order reaction. While the Escherichia coli enzyme lost more than 90% of its activity after 5 min
at 70³C, the activity of T. thermophilus GDH was not a¡ected by this treatment. The puri¢ed enzyme showed a half-life ranging from approximately 58 h at 72³C (the optimum growth temperature) to 5 h at 80³C and 30 min at 85³C. In contrast to other enzymes from hyperthermophilic microorganisms that show a relatively short inactivation half-life at their respective growth optimum temperatures, [10], GDH from T. thermophilus has a relatively long half-life (58 h at 72³C). The temperature dependence of parameters for the inactivation reaction was determined from this plot. We obtained the following
Fig. 2. Arrhenius plot for temperature dependence of T. thermophilus glutamate dehydrogenase (deamination reaction).
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Fig. 3. Arrhenius plot of the inactivation reaction of NAD-glutamate dehydrogenase from T. thermophilus. For each temperature, ¢rst order rate constants (k) were determined from plots of ln(% activity remaining) versus time of incubation, and plotted as ln(k) versus 1/temperature (³K).
values: 383 kJ mol31 and 394 kJ mol31 for the activation energy (Eadenat ) and enthalpy (vHdenat ) of the denaturation process, respectively (Fig. 3). According to calorimetric experiments thermal denaturation occurs at approximately 95³C, and is an irreversible process as demonstrated by the absence of any calorimetric peak in the second scanning of the same sample. Side reactions occurring at high temperatures and aggregation phenomena probably prevent the correct refolding of polypeptide chains [11]. 4. Discussion In this study, we puri¢ed to homogeneity and characterised an NAD-speci¢c glutamate dehydrogenase from T. thermophilus. As can be seen in SDS-PAGE, the enzyme migrated as a double band (50 and 48 kDa). Multiple bands may be caused by several modi¢cations such as proteolysis during the preparation or may be due to artifacts in SDSPAGE. Bacterial and fungal NADP-speci¢c and vertebrate dual-speci¢city GDHs have a hexameric structure (Mr 48^53.5 kDa) [1], and NAD-speci¢c enzymes have either four identical subunits (Mr 115 kDa), as found in Neurospora crassa [2], or six identical subunits (Mr 48 kDa), as found in Clostridium symbiosum and Peptostreptococcus asaccharolyticus
19
[12]. Therefore, T. thermophilus GDH seems to be a member of the group of hexameric GDHs. The thermostable GDH serves a catabolic function by catalysing the degradation of glutamate to 2-oxoglutarate. This conclusion is based on the absence of activity in the reductive amination direction. The apparent Km value for the NAD substrate is of the same order of magnitude as that reported for Halobacterium salinarum GDH (0.3 mM) [13], Bacillus cereus (0.56 mM) [14] and the cyanobacterium Synechocystis (0.56 mM) [15]. However, the Km value for L-glutamate is considered high in comparison with H. salinarum (11 mM) [13], Bacillus cereus (7.7 mM) [14] and the cyanobacterium Synechocystis (16.1 mM) [15]. The T. thermophilus enzyme shows maximum activity at 85^90³C, slightly higher than the growth temperature; in this respect the enzyme is similar to that of Thermococcus litoralis [16]. A linear dependence of ln(Vmax ) on 1/T was found over the temperature range tested. In contrast, non-linear Arrhenius plots have been described for several enzymes from thermophilic Archaea and Bacteria, and interpreted as indicative of temperatureinduced conformational changes [10]. The activation energy calculated from the Arrhenius plot was 36 kJ mol31 , lower than that calculated for the enzymes from Proteus inconstans (54 kJ mol31 ) and Thermococcus profundus (45.3 kJ mol31 ) [17]. Di¡erent activation energies could re£ect a di¡erent conformational £exibility of the enzyme depending on temperature increase [18]. T. thermophilus GDH is thermoactive [19], a property shared with enzymes from hyperthermophiles and mesophiles. Almost all enzymes show temperature optima at or slightly above the growth temperature of the source organism [19]. The enzyme from T. thermophilus is not as thermostable as those of P. furiosus and ES4 or T. litoralis. For example, P. furiosus and ES4 GDHs have an apparent half-life of about 10 h at 100³C, T. litoralis GDH retained 50% of its activity after 2 h at 98³C, whereas T. thermophilus GDH has a halflife of 30 min at 85³C. DiRuggiero and Robb [20] suggested that there is a correlation between growth temperature and enzyme thermostability. T. thermophilus GDH did not show a dual cofactor speci¢city. Such a duality is rarely found in Bacteria but is common in thermophilic and hyperthermophilic Archaea and vertebrate Eukarya. In
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addition, the GDH of T. thermophilus showed no NADH-dependent reductive amination of 2-oxoglutarate, in contrast to the Thermotoga maritima GDH [21], which showed all four GDH activities, i.e. reductive amination of 2-oxoglutarate with NADH and NADPH, as well as oxidative deamination of L-glutamate with NAD and NADP as the cosubstrates. This suggests that GDH from T. thermophilus has a catabolic function in contrast to T. maritima GDH [21], which has an anabolic function.
Acknowledgments We thank Dra. Mar|èa Joseè Munìoz for her kind help in the calorimetric experiments. Financial support from the Generalitat Valenciana (research grant to M.J.B.) and from the Instituto de Cultura Juan Gil-Albert (fellowship to J.L.R.) is gratefully acknowledged.
References [1] Smith, E.L., Austen, B.M., Blumenthal, K.M. and Nyc, J.F. (1975) Glutamate dehydrogenases. In: The Enzymes (Boyer, P.D., Ed.), Vol. 11, 3rd edn., pp. 294^367. Academic Press, New York. [2] Veronese, F.M., Nyc, J.F., Degani, Y., Brown, D.M. and Smith, E.L. (1974) Nicotinamide adenine dinucleotide-speci¢c glutamate dehydrogenase of Neurospora. J. Biol. Chem. 249, 7922^7928. [3] Bonete, M.J., Camacho, M.L. and Cadenas, E. (1987) A new glutamate dehydrogenase from Halobacterium halobium with di¡erent coenzyme speci¢city. Int. J. Biochem. 19, 1149^1155. [4] Ferrer, J., Peèrez-Pomares, F. and Bonete, M.J. (1996) NADPglutamate dehydrogenase from the halophilic archaeon Haloferax mediterranei: enzyme puri¢cation, N-terminal sequence and stability. FEMS Microbiol. Lett. 141, 59^63. [5] Oshima, T. and Imahori, K. (1974) Description of Thermus thermophilus (Yoshida and Oshima), comb. nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa. Int. J. Syst. Bacteriol. 24, 102^112. [6] Hartmann, R.K., Wolters, J., Kroëger, B., Schultze, S., Specht, T. and Erdmann, V.A. (1989) Does Thermus represent another deep eubacterial branching ? Syst. Appl. Microbiol. 11, 243^ 249. [7] Lasa, I. and Berenguer, J. (1993) Thermophilic enzymes and their biotechnological potential. Microbiologia SEM 9, 77^89. [8] Cleland, W.W. (1979) Statistical analysis of enzyme kinetic data. Methods Enzymol. 63, 103^138.
[9] Hornby, D.P. and Engel, P.C. (1984) Characterisation of Peptostreptococcus asaccharolyticus glutamate dehydrogenase puri¢ed by dye-ligand chromatography. J. Gen. Microbiol. 130, 2385^2394. [10] Hess, D., Kruëger, K., Knappik, A., Palm, P. and Hensel, R. (1995) Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea. Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein. Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus. Eur. J. Biochem. 233, 227^ 237. [11] D'Auria, S., Rossi, M., Barone, G., Catanzano, F., Del Vecchio, Graziano, G. and Nucci, R. (1996) Temperature-induced denaturation of L-glycosidase from the Archaeon Sulfolobus solfataricus. J. Biochem. 120, 292^300. [12] Lilley, K.S., Baker, P.J., Britton, K.L., Stillman, T.J., Brown, P.E., Moir, A.J.G., Engel, P.C., Rice, D.W., Bell, J.E. and Bell, E. (1991) The partial amino acid sequence of the NAD-dependent glutamate dehydrogenase of Clostridium symbiosum : implications for the evolution and structural basis of coenzyme speci¢city. Biochim. Biophys. Acta 1080, 191^ 197. [13] Bonete, M.J., Camacho, M.L. and Cadenas, E. (1989) Kinetic mechanism of Halobacterium halobium NAD -glutamate dehydrogenase. Biochim. Biophys. Acta 990, 150^155. [14] Jahns, T. and Kaltwasser, H. (1993) Properties of the coldlabile NAD -speci¢c glutamate dehydrogenase from Bacillus cereus DSM 31. J. Gen. Microbiol. 139, 775^780. [15] Chavez, S. and Candau, P. (1991) An NAD-speci¢c glutamate dehydrogenase from cyanobacteria. Identi¢cation and properties. FEBS Lett. 285, 35^38. [16] Ohshima, T. and Nishida, N. (1994) Puri¢cation and characterization of extremely thermostable glutamate dehydrogenase from a hyperthermophilic Archaeon, Thermococcus litoralis. Biocatalysis 11, 117^129. [17] Kobayashi, T., Higuchi, S., Kimura, K., Kudo, T. and Horikoshi, K. (1995) Properties of glutamate dehydrogenase and its involvement in alanine production in a hyperthermophilic Archaeon, Thermococcus profundus. J. Biochem. 118, 587^592. [18] Wrba, A., Schweiger, A., Schultes, V. and Jaenicke, R. (1990) Extremely thermostable D-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. Biochemistry 29, 7584^7592. [19] Danson, M.J., Hough, D.W., Russell, R.J.M., Taylor, G.L. and Pearl, L. (1996) Enzyme thermostability and thermoactivity. Protein Eng. 9, 629^630. [20] DiRuggiero, J. and Robb, F.T. (1996) Enzymes of central nitrogen metabolism from hyperthermophiles. Characterization, thermostability, and genetics. Adv. Protein Chem. 18, 311^339. [21] Kort, R., Liebl, W., Labedan, B., Forterre, P., Eggen, R.I.L. and de Vos, W.M. (1997) Glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima: molecular characterization and phylogenetic implications. Extremophiles 1, 52^60.
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NAD-speci¢c glutamate dehydrogenase from Thermus thermophilus HB8: puri¢cation and enzymatic properties Joseè Luis Ruiz, Juan Ferrer, Moènica Camacho, Mar|èa Joseè Bonete * Divisioèn de Bioqu|èmica, Facultad de Ciencias, Universidad de Alicante, Ap. 99, 03080 Alicante, Spain Received 27 August 1997; revised 25 November 1997; accepted 26 November 1997
Abstract An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85^90³C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented Km values for NAD and L-glutamate of 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72³C. Differential scanning calorimetry revealed a tm of 95³C for denaturation. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : NAD-glutamate dehydrogenase ; Thermus thermophilus; Thermostability
1. Introduction Glutamate dehydrogenases (L-glutamate NAD(P) oxidoreductase; GDHs, EC 1.4.1.2^4) have been isolated from a variety of species. The enzymes catalyse the reversible oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia using NAD or NADP as cofactors [1] according to the following reaction: l3Glutamate NAD P H2 OH 23oxoglutarate NH 4 NAD PH H
Glutamate dehydrogenases are important enzymes in * Corresponding author. Tel.: +34 (6) 590 3524; Fax: +34 (6) 590 3464; E-mail: [email protected]
the metabolism of most organisms since they provide a link between carbon and nitrogen metabolism [1]. They are involved in ammonia assimilation and catabolism of amino acids in a wide range of organisms. Three di¡erent glutamate dehydrogenases have been described: an NAD-speci¢c enzyme (EC 1.4.1.2), an NADP-speci¢c enzyme (EC 1.4.1.4) and an NAD(P)-dependent enzyme (EC 1.4.1.3). In general, prokaryotic glutamate dehydrogenase enzymes are speci¢c for either NAD in the case of catabolic enzymes or NADP in the case of biosynthetic GDHs. Enzymes puri¢ed from vertebrates can use both coenzymes. Fungi and some halophiles have two distinct glutamate dehydrogenases with kinetic properties that allow them to have only one physiological role [2^4]. Glutamate dehydrogenase is evolutionarily conserved in all three primary domains:
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Eucarya, Bacteria and Archaea, and information is available on structural, enzymatic and phylogenetic properties. We examined the activity of glutamate dehydrogenase in the cell homogenate of Thermus thermophilus, a Gram-negative aerobic microorganism, which can grow at temperatures above 75³C, and is one of the most widely studied thermophilic Bacteria [5]. This extreme thermophile belongs to one of the oldest phylogenetic branches of the Bacteria domain [6]. Cellular components of members of the genus Thermus are generally more resistant to heat and most common protein denaturants than their counterparts among mesophilic bacteria, which makes them extremely interesting for industrial processes. The genus Thermus is receiving considerable attention for its biotechnological potential [7]. Therefore, GDH from this microorganism is an attractive choice to study evolutionary aspects of enzyme structure, function and stability. In this report, we describe the puri¢cation and biochemical characterisation of the NAD-dependent glutamate dehydrogenase from T. thermophilus.
2. Materials and methods 2.1. Organisms T. thermophilus HB8 cells were kindly supplied by Dr. Joseè Berenguer from Centro Nacional de Biotecnolog|èa `Severo Ochoa' (Madrid). 2.2. Enzyme puri¢cation Frozen cells of T. thermophilus (30 g) were suspended in 150 ml of 200 mM Tris-HCl bu¡er (pH 8) containing 2 mM EDTA and 2 mM 2-mercaptoethanol to preserve GDH activity during storage and 1 mM phenylmethylsulfonyl £uoride (PMSF), a serine protease inhibitor, to prevent proteolysis. Cells were disrupted by mechanical and sonic treatment. Cell debris were removed by centrifugation at 12 000Ug at 4³C for 30 min. The supernatant was centrifuged at 105 000Ug for 2 h. The new supernatant was used for enzyme puri¢cation. With the exception of (NH4 )2 SO4 precipitation, all puri¢cation steps were carried out at room temperature.
Step 1: Solid (NH4 )2 SO4 was added to crude extract with mild stirring at 4³C up to 35% saturation. Stirring was continued for 45 min and the precipitate was collected and dissolved in 50 mM Tris-HCl buffer (pH 8), containing 2 mM EDTA and 2 mM 2mercaptoethanol (TM bu¡er). Step 2: After desalting through dialysis against TM bu¡er, the enzyme solution was loaded onto an anion-exchange column (5U2.5 cm) of Q-Sepharose (Pharmacia) equilibrated with TM bu¡er. The enzyme was eluted with a 200-ml linear gradient of 0^1 M NaCl in TM bu¡er. Fractions containing enzyme activity were pooled and dialysed against TM bu¡er. Step 3: The clear solution was applied onto a Blue-Sepharose CL-6B column (7U2.5 cm), equilibrated and washed with TM bu¡er. The enzyme was eluted with the same bu¡er containing 3 mM NAD. The active fractions were pooled. Step 4: The solution of the previous step was applied onto the Q-Sepharose column as in step 2. Step 5: Finally, the high-speci¢c-activity material from step 4 was applied onto a column (64U2.5 cm) of G-100 Sephadex (Pharmacia) equilibrated with TM bu¡er (pH 8). The enzyme was eluted from the column with the same bu¡er. The most active fractions were pooled. 2.3. Assay of glutamate dehydrogenase and protein determinations The activity of GDH was assayed spectrophotometrically at 340 nm and 72³C in 50 mM Tris-HCl bu¡er (pH 8), 2 mM EDTA containing 100 mM Lglutamate and 3 mM NAD. After equilibration at 72³C for 5 min, the reaction was started by the addition of NAD. One unit of enzyme activity is the reduction of 1 Wmol of NAD per minute. The values of Vmax and Km were determined using Cleland programs [8]. Protein concentrations were determined applying the method of Bradford. 2.4. Determination of native Mr values The Mr for the native glutamate dehydrogenase was estimated by gel ¢ltration on a Sepharose CL6B (40U2.5 cm) column. The ¢ltration was carried out using a 50 mM Tris-HCl bu¡er (pH 8) contain-
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ing 2 mM EDTA and 2 mM 2-mercaptoethanol for the standard protein markers and the Thermus glutamate dehydrogenase. Protein markers were: bovine glutamate dehydrogenase (330 kDa), catalase (240 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa) and cytochrome c (12.5 kDa). 2.5. Electrophoresis The subunit Mr of the puri¢ed glutamate dehydrogenase was determined by SDS-PAGE. Native PAGE was carried out at pH 8.9 on a 7.5% separation gel. Glutamate dehydrogenase was visualised by coupling the glutamate-dependent reduction of NAD with diaformazan production. After electrophoresis, gels were incubated in the dark for 10 min at 72³C in a solution of 50 mM Tris-HCl (pH 8) containing 100 mM L-glutamate, 3 mM NAD, 0.05% (w/v) nitroblue tetrazolium (Sigma) and 0.005% (w/v) phenazine methosulfate (Sigma), followed by ¢xing in ethanol, acetic acid, glycerol and water (5:2:1:4) for storage. 2.6. Di¡erential scanning calorimetry Scanning calorimetric measurements were carried out on a Setaram DSC92 scanning microcalorimeter at a heating rate of 1³K min31 . All measurements were performed under a helium atmosphere at a total pressure of 3 bar. Protein samples were extensively dialysed against 50 mM Tris-HCl bu¡er pH 8.0. The protein concentration in these experiments was 1.4 mg ml31 . The bu¡er-bu¡er baseline was obtained at the same scanning rate and was subtracted from the sample curve.
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3. Results The result of a typical puri¢cation procedure is summarised in Table 1. Cell-free extracts of T. thermophilus contained signi¢cant amounts of GDH activity. The extract was very stable at 8³C and 23³C; no activity was lost even after 36 days if the extract was maintained in 200 mM Tris-HCl bu¡er (pH 8) containing 2 mM EDTA, 2 mM 2-mercaptoethanol and 1 mM PMSF. The elution of the NAD-GDH from the anion-exchange column, followed by direct application onto the Blue-Sepharose CL-6B column, was crucial for the puri¢cation procedure. Triazine dyes have proved useful for the isolation of NADdependent enzymes [9]. The ¢nal preparation had a speci¢c activity of 70 U mg31 and the enzyme was puri¢ed 350-fold. The thermophilic enzyme was found to be homogeneous as judged by electrophoresis on SDS-PAGE. We also carried out analytical polyacrylamide gel electrophoresis. The enzyme migrated as a single component, and only one band of GDH activity was found through activity staining. The enzymatic activity corresponded to the stained protein band obtained with Coomassie staining. Electrophoresis of the puri¢ed GDH from T. thermophilus with anionic (SDS) detergent revealed that the enzyme migrated as a double protein band (Fig. 1). The mobility of the enzyme in SDS-PAGE corresponded to subunits Mr of 50 þ 3 kDa and 48 þ 3 kDa. An apparent molecular mass of 289 þ 30 kDa was found for the puri¢ed GDH, as estimated by gel ¢ltration, so we can conclude that glutamate dehydrogenase from T. thermophilus consists of six subunits. The enzyme speci¢cally required NAD and L-glutamate as the coenzyme and substrate respectively for the deaminating activity. T. thermophilus GDH is catalytically active only with NAD as a coenzyme
Table 1 Puri¢cation of NAD-glutamate dehydrogenase from T. thermophilus Step
Enzyme activity (U)
Total protein (mg)
Speci¢c activity (U mg31 )
Puri¢cation (n-fold)
Yield (%)
Crude extract Ammonium sulfate 35% Q-Sepharose Blue-Sepharose Q-Sepharose Sephadex G-100
250 145 110 36 30 14
1473 168 32 1 0.54 0.20
0.2 0.9 3.4 36 56 70
1 5 17 180 275 350
100 59 44 14 12 6
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Fig. 1. SDS-PAGE of T. thermophilus glutamate dehydrogenase. Lane 1, 5 Wg of protein markers (middle range of Promega) ; lane 2, 3 Wg of pooled fractions after Sephadex G-100.
and it could not be replaced by NADP. Kinetic parameters for the deamination oxidative reaction were determined under conditions of 72³C and pH 8 (in TM bu¡er). The Km values for NAD and L-glutamate were 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. The amino acids L-leucine, L-valine and L-isoleucine were tested as substrates, but were not oxidised by GDH. On the other hand, no activity in the direction of L-glutamate biosynthesis (amination of 2-oxoglutarate) was detected. The dependence on pH of the puri¢ed GDH activity was examined at 72³C using di¡erent bu¡ers ranging from pH 5 to pH 10. All pH values were corrected as regards the temperature e¡ect on pH. Optimal activity occurred in 50 mM Tris-HCl bu¡er pH 8.0, containing 2 mM EDTA. The temperature dependence of Vmax (deamination reaction) was determined over 35^95³C and documented as an Arrhenius plot (Fig. 2). Optimal activity was observed at 85^90³C. Taking into account the temperature activity dependence, the activation energy (Ea ) for the oxidative deamination of L-glutamate was found to be 36 kJ mol31 . Thermal inactivation kinetics were measured at di¡erent temperatures. Heat-induced inactivation kinetics of the enzyme ¢t very well into an irreversible ¢rst-order reaction. While the Escherichia coli enzyme lost more than 90% of its activity after 5 min
at 70³C, the activity of T. thermophilus GDH was not a¡ected by this treatment. The puri¢ed enzyme showed a half-life ranging from approximately 58 h at 72³C (the optimum growth temperature) to 5 h at 80³C and 30 min at 85³C. In contrast to other enzymes from hyperthermophilic microorganisms that show a relatively short inactivation half-life at their respective growth optimum temperatures, [10], GDH from T. thermophilus has a relatively long half-life (58 h at 72³C). The temperature dependence of parameters for the inactivation reaction was determined from this plot. We obtained the following
Fig. 2. Arrhenius plot for temperature dependence of T. thermophilus glutamate dehydrogenase (deamination reaction).
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Fig. 3. Arrhenius plot of the inactivation reaction of NAD-glutamate dehydrogenase from T. thermophilus. For each temperature, ¢rst order rate constants (k) were determined from plots of ln(% activity remaining) versus time of incubation, and plotted as ln(k) versus 1/temperature (³K).
values: 383 kJ mol31 and 394 kJ mol31 for the activation energy (Eadenat ) and enthalpy (vHdenat ) of the denaturation process, respectively (Fig. 3). According to calorimetric experiments thermal denaturation occurs at approximately 95³C, and is an irreversible process as demonstrated by the absence of any calorimetric peak in the second scanning of the same sample. Side reactions occurring at high temperatures and aggregation phenomena probably prevent the correct refolding of polypeptide chains [11]. 4. Discussion In this study, we puri¢ed to homogeneity and characterised an NAD-speci¢c glutamate dehydrogenase from T. thermophilus. As can be seen in SDS-PAGE, the enzyme migrated as a double band (50 and 48 kDa). Multiple bands may be caused by several modi¢cations such as proteolysis during the preparation or may be due to artifacts in SDSPAGE. Bacterial and fungal NADP-speci¢c and vertebrate dual-speci¢city GDHs have a hexameric structure (Mr 48^53.5 kDa) [1], and NAD-speci¢c enzymes have either four identical subunits (Mr 115 kDa), as found in Neurospora crassa [2], or six identical subunits (Mr 48 kDa), as found in Clostridium symbiosum and Peptostreptococcus asaccharolyticus
19
[12]. Therefore, T. thermophilus GDH seems to be a member of the group of hexameric GDHs. The thermostable GDH serves a catabolic function by catalysing the degradation of glutamate to 2-oxoglutarate. This conclusion is based on the absence of activity in the reductive amination direction. The apparent Km value for the NAD substrate is of the same order of magnitude as that reported for Halobacterium salinarum GDH (0.3 mM) [13], Bacillus cereus (0.56 mM) [14] and the cyanobacterium Synechocystis (0.56 mM) [15]. However, the Km value for L-glutamate is considered high in comparison with H. salinarum (11 mM) [13], Bacillus cereus (7.7 mM) [14] and the cyanobacterium Synechocystis (16.1 mM) [15]. The T. thermophilus enzyme shows maximum activity at 85^90³C, slightly higher than the growth temperature; in this respect the enzyme is similar to that of Thermococcus litoralis [16]. A linear dependence of ln(Vmax ) on 1/T was found over the temperature range tested. In contrast, non-linear Arrhenius plots have been described for several enzymes from thermophilic Archaea and Bacteria, and interpreted as indicative of temperatureinduced conformational changes [10]. The activation energy calculated from the Arrhenius plot was 36 kJ mol31 , lower than that calculated for the enzymes from Proteus inconstans (54 kJ mol31 ) and Thermococcus profundus (45.3 kJ mol31 ) [17]. Di¡erent activation energies could re£ect a di¡erent conformational £exibility of the enzyme depending on temperature increase [18]. T. thermophilus GDH is thermoactive [19], a property shared with enzymes from hyperthermophiles and mesophiles. Almost all enzymes show temperature optima at or slightly above the growth temperature of the source organism [19]. The enzyme from T. thermophilus is not as thermostable as those of P. furiosus and ES4 or T. litoralis. For example, P. furiosus and ES4 GDHs have an apparent half-life of about 10 h at 100³C, T. litoralis GDH retained 50% of its activity after 2 h at 98³C, whereas T. thermophilus GDH has a halflife of 30 min at 85³C. DiRuggiero and Robb [20] suggested that there is a correlation between growth temperature and enzyme thermostability. T. thermophilus GDH did not show a dual cofactor speci¢city. Such a duality is rarely found in Bacteria but is common in thermophilic and hyperthermophilic Archaea and vertebrate Eukarya. In
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addition, the GDH of T. thermophilus showed no NADH-dependent reductive amination of 2-oxoglutarate, in contrast to the Thermotoga maritima GDH [21], which showed all four GDH activities, i.e. reductive amination of 2-oxoglutarate with NADH and NADPH, as well as oxidative deamination of L-glutamate with NAD and NADP as the cosubstrates. This suggests that GDH from T. thermophilus has a catabolic function in contrast to T. maritima GDH [21], which has an anabolic function.
Acknowledgments We thank Dra. Mar|èa Joseè Munìoz for her kind help in the calorimetric experiments. Financial support from the Generalitat Valenciana (research grant to M.J.B.) and from the Instituto de Cultura Juan Gil-Albert (fellowship to J.L.R.) is gratefully acknowledged.
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