Comparative Analysis of Pyruvate Kinases from the Hyperthermophilic Archaea Archaeoglobus fulgidus, Aeropyrum pernix, and Pyrobaculum aerophilum and the Hyperthermophilic Bacterium Thermotoga maritima

UNUSUAL REGULATORY PROPERTIES IN HYPERTHERMOPHILIC ARCHAEA*

Ulrike Johnsen, Thomas Hansen and Peter Schönheit {ddagger}

From the Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1–9, Kiel D-24118, Germany

Received for publication, October 8, 2002 , and in revised form, March 21, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyruvate kinases (PK, EC 2.7.1.40 [EC] ) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 °C (A. fulgidus) and above 98 °C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 °C (A. pernix) and showed melting temperatures for thermal unfolding at 93 °C (A. fulgidus) or above 98 °C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 °C, was highly thermostable up to 90 °C, and had a melting temperature above 98 °C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperthermophilic prokaryotes, with an optimal growth temperature higher than 80 °C, are considered to represent the phylogenetically most ancestral organisms (1). Recent comparative studies of the hexose degradation pathways in hyperthermophilic archaea and in the hyperthermophilic bacterium Thermotoga revealed that the classic Embden-Meyerhof (EM)1 pathway is operative only in Thermotoga, whereas in all archaea, the EM pathway exists in modified versions. The modified EM pathways contain, e.g. unusual glucokinases (GLK) and 6-phosphofructokinases (PFK) such as ADP-dependent GLK and ADP-dependent PFK in Pyrococcus, Thermococcus, and Archaeoglobus; unusual ATP-dependent archaeal GLKs of the ROK (Regulators, ORFs, Kinases) protein family; non-regulatory ATP-dependent PFKs in Desulfurococcus and Aeropyrum; and pyrophosphate-dependent PFK in Thermoproteus. In addition, the modified EM pathways contain novel enzymes of glyceraldehyde 3-phosphate (GAP) oxidation to 3-phosphoglycerate, such as GAP:ferredoxin oxidoreductase and non-phosphorylative glyceraldehyde-3-phosphate dehydrogenase, which replace GAP dehydrogenase and phosphoglycerate kinase in the conventional EM pathway (26).

An important regulatory principle of the carbon flux in the classic EM pathway of eukarya and bacteria is the allosteric regulation of two key enzymes, ATP-dependent PFK and pyruvate kinase. Both enzymes are considered to catalyze irreversible reactions in vivo and have been shown to be allosterically activated or inhibited by intermediates of metabolism or by the energy charge of the cell. To get insights into the role of allosteric regulation of the modified EM pathways, the PFKs of various hyperthermophilic archaea have been characterized. It was found that all archaeal PFKs (ADP-, ATP-, and pyrophosphate-dependent) were not allosterically regulated by classic effectors of ATP-PFKs of eukarya and bacteria, such as ADP and PEP (711). Thus, PFKs appear not to be a site of allosteric control in the modified EM pathways of hyperthermophilic archaea. In contrast, ATP-PFK from the hyperthermophilic bacterium Thermotoga maritima shows the classic response toward the allosteric effectors, it was activated by ADP and inhibited by PEP (12). Thus, the ATP-PFK of Thermotoga represents a site of allosteric control in the conventional EM pathway operative under hyperthermophilic conditions. To identify potential allosteric sites of the modified EM pathways of hyperthermophilic archaea, we studied the regulatory properties of pyruvate kinases, which catalyze the irreversible conversion of PEP to pyruvate, the terminal reaction of both modified and conventional EM pathways.

PKs are well characterized enzymes from many eukarya and bacteria (1315). The PKs are usually homotetrameric enzymes of about 200 kDa composed of 50-kDa subunits; the enzymes require divalent cations for activity; many PKs were shown to be activated by monovalent cations, K+ or . With a few exceptions, all PKs from eukarya and bacteria are allosterically regulated either by intermediates of sugar metabolism, usually sugar phosphates, or by adenosine nucleotides reflecting the energy charge of the cells. Most eukaryal PKs are allosterically activated by fructose 1,6-bisphosphate (FBP). An unusual allosteric effector, fructose 2,6-bisphosphate, has recently been reported for the PKs of the protozoa Leishmania mexicana and Trypanosoma brucei (16, 17). Several bacterial PKs are activated by FBP, but the majority of bacterial PKs show allosteric activation by AMP and sugar monophosphates (e.g. ribose 5-phosphate). Few bacteria, e.g. Escherichia coli and Salmonella typhimurium contain two PK isoenzymes being activated either by FBP or by AMP (1820). Few nonallosteric PKs have also been described, e.g. M1 isoenzyme of vertebrates and the dimeric PKs from Schizosaccharomyces pombe and Zymomonas mobilis (15, 21, 22). More than 140 primary sequences of eukarya and bacteria are known. These include putative PK homologs found in all available archaeal genomes with the exception of the methanogens Methanopyrus kandleri and Methanothermobacter thermoautotrophicus and the hyperthermophilic sulfate reducer Archaeoglobus fulgidus VC16 (35). Interestingly, the closely related strain A. fulgidus 7324 contains high pyruvate kinase activity as part of a modified EM pathway (3). Crystal structures of PKs are available for the enzymes from cat and rabbit muscle, yeast, and E. coli and of non-allosteric M1 PK isoenzymes from vertebrates (2427). The binding sites for PEP and for the allosteric activator FBP from the yeast PK were identified.

To date, only two PKs from the domain of archaea have been biochemically characterized, from the hyperthermophile Thermoproteus tenax and from the moderate thermophile Thermoplasma acidophilum. Both enzymes are homotetrameric proteins. For the T. tenax PK a response to heterotropic allosteric effectors was not found, whereas the PK from T. acidophilum has been shown to be activated by AMP (28, 29).

In this communication we performed a comparative study on PKs, from three hyperthermophilic archaea, the crenarchaeota Aeropyrum pernix and Pyrobaculum aerophilum as well as the euryarchaeon A. fulgidus strain 7324 and from the hyperthermophilic bacterium T. maritima. The thermophilic, kinetic, and, in particular, the regulatory properties as well as the phylogenetic affiliation of the PKs were analyzed. It was found that all PKs from hyperthermophilic archaea and bacteria are homotetrameric proteins of extreme thermostability showing temperature optima up to 100 °C for catalytic activity. An unusual property of PKs from all hyperthermophilic archaea was the absence of regulation by classic heterotropic effectors. In contrast, the PK from the hyperthermophilic bacterium Thermotoga showed the classic response to allosteric effectors. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Growth of A. fulgidus and T. maritima and Preparation of Cell Extracts—A. fulgidus strain 7324 and T. maritima were grown anaerobically in the presence of starch as described (3, 3032). Cells were harvested in the late exponential growth phase. Cell extracts were prepared from 80 g (A. fulgidus) and 60 g (T. maritima) of frozen cells, which were suspended in 150 ml of 50 mM Tris-HCl, pH 7.0, containing 10 mM NaCl and 1 mM dithioerythritol and in 90 ml of 50 mM Tris-HCl, pH 7.0, containing 2 mM dithioerythritol, respectively. Cells were disrupted by passing through a French pressure cell at 1.3 x 108 Pa. Cell debris and unbroken cells were removed by centrifugation for 90 min at 100,000 x g at 4 °C.

Purification of PK from A. fulgidus—The 100,000 x g supernatant was applied to a Q-Sepharose HiLoad column (22 x 5 cm), which had been equilibrated with buffer A (50 mM Tris-HCl, pH 9.0, 1 mM dithioerythritol). Protein was eluted with a decreasing pH gradient from 9.0 to 7.0 in buffer A and from pH 7.0 to 6.5 in 50 mM bis-Tris-propane, pH 6.5, containing 1 mM dithioerythritol. Fractions containing the highest PK activity were pooled and, after pH exchange (pH 9.0), applied to a Uno-Q5 column (5 ml), equilibrated with buffer A. Protein was desorbed with a NaCl gradient from 0 to 0.5 M in buffer A. Fractions containing the highest PK activity were pooled and concentrated to a volume of 1 ml by ultrafiltration. The concentrated protein solution was applied to a Superdex 200 HiLoad 16/60 gel filtration column equilibrated with 50 mM MES, pH 6.5, containing 50 mM NaCl and 1 mM dithioerythritol. Eluted PK-containing fractions were pooled and applied to a Uno-S1 column (1 ml), equilibrated with 50 mM acetate, pH 5.3, containing 1 mM dithioerythritol. Protein was eluted with a linear NaCl gradient of 0 to 1 M. Pure enzyme was eluted at 0.25 M NaCl.

Purification of PK from T. maritima—The 100,000 x g supernatant was applied to a Q-Sepharose HiLoad column (22 x 5 cm), which had been equilibrated with 50 mM Tris-HCl, pH 7.0, containing 2 mM dithioerythritol. After washing the column with 50 mM piperazine, pH 6.4, containing 2 mM dithioerythritol (buffer B), protein was eluted with an increasing gradient from 0 to 2 M NaCl in buffer B. Fractions containing the highest PK activity (0.15–0.3 M NaCl) were pooled and applied to an SP-Sepharose column (75 ml) equilibrated with 50 mM MES, pH 5.5, containing 2 mM dithioerythritol. Protein was desorbed with a pH gradient from pH 5.5 to 7.5 in 50 mM MES. Fractions containing the highest PK activity were pooled and concentrated to a volume of 1 ml by ultrafiltration (exclusion size, 20 kDa). The protein solution was applied to a Superdex 200 HiLoad 16/60 gel filtration column equilibrated with 50 mM Tris-HCl, pH 7.0, containing 50 mM NaCl and 2 mM dithioerythritol. The eluted PK activity-containing fractions were pooled and applied to a Uno-S1 column (1 ml) equilibrated with 50 mM MES, pH 5.5, containing 2 mM dithioerythritol. Protein was eluted with a linear gradient of 0 to 1 M NaCl. Fractions containing the highest PK activity were pooled and applied to a Uno-Q1 column (1 ml) equilibrated with 50 mM Tris-HCl, pH 8.0, containing 1 mM dithioerythritol. Protein was eluted with a gradient of pH 8.0 to 5.3 (50 mM piperazine, 2 mM dithioerythritol). The fraction containing the highest PK activity was eluted at pH 5.5, yielding pure enzyme.

Cloning and Functional Overexpression of ORF TM0208 Coding for PK of T. maritima and Purification of Recombinant Enzyme—ORF TM0208 was amplified by PCR. The PCR product was cloned into pET19b via two restriction sites (NdeI and BamHI) created with the primers 5'-CGGGGTGAACATATGCGAAGTACAAAGAT-3' and 5'-ATCTTCATAGGGATCCCCCCTCAATCCA-3'. The vector pET19b(pyk-TM0208) was transformed into E. coli BL21 codon plus(DE3)-RIL cells. For expression, cells were grown in Luria-Bertani medium at 37 °C. The expression was started by inducing the promoter with isopropyl-1-thio-{beta}-D-galactopyranoside. After3hof further growth, cells were harvested by centrifugation. The pellet was suspended in buffer C (20 mM Tris-HCl, pH 8.2, containing 0.3 M NaCl and 4 mM imidazole). Cells were disrupted through a French pressure cell. After centrifugation (48,000 x g, 4 °C, 30 min), the supernatant was heat-precipitated at 70 °C for 30 min, followed by an additional centrifugation step (100,000 x g, 4 °C, 60 min). The heat-precipitated supernatant was applied to a Ni-NTA column (7 ml) equilibrated with buffer C. Protein was eluted with increasing imidazole concentration from 4 to 500 mM in buffer C. Fractions containing the highest enzyme activity were pooled and concentrated to a volume of 1 ml by ultrafiltration. The protein solution was applied to a Superdex 200 HiLoad 16/60 gel filtration column equilibrated with 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl and 1 mM dithioerythritol. Protein was eluted. Fractions containing the highest PK activity were pooled and applied to a Uno-S1 column (1 ml) equilibrated with 50 mM MES, pH 5.5, containing 1 mM dithioerythritol. Protein was eluted with a gradient of 0 to 1 M NaCl. The fraction containing the PK activity was recovered at 0.8 M NaCl. At this stage PK was essentially pure.

Cloning and Functional Overexpression of ORF APE0489 Coding for PK of A. pernix and Purification of Recombinant Enzyme—ORF APE0489 was amplified and cloned into pET19b via two restriction sites (NdeI and EcoR1) created with the primers 5'-TTAGAGAGGCTGGCCTCATATGAGGGG-3' and 5'-GATAGGAATTCAGACAGGAGCGGCTAG-3'. The vector pET19b(pyk-APE0489) was transformed into E. coli BL21 codon plus(DE3)-RIL cells. The expression and cell harvesting was performed as described above. The pellet was resuspended in buffer C. Cells were disrupted by passing through a French pressure cell. After centrifugation, the supernatant was heat-precipitated at 77 °C for 30 min and centrifuged (100,000 x g, 4 °C for 60 min) again. The supernatant was applied to a Ni-NTA column (7 ml) equilibrated with buffer C. Protein was eluted with an imidazole gradient from 4 to 500 mM in buffer C. Fractions containing the highest PK activity were pooled, incubated at 100 °C for 15 min, and centrifuged. At this stage PK was essentially pure.

Cloning and Functional Overexpression of ORF PAE0819 Coding for PK of P. aerophilum and Purification of Recombinant Enzyme—ORF PAE0819 was amplified by PCR and was cloned into pET17b via two restriction sites (NdeI and BamH1) created with the primers 5'-CACTAAAGGGCGCGGACATATGAGCGCTC-3' and 5'-GTTGGGTACGCCAGGATCCTCTTTTACCG-3'. The vector pET17b(pyk-PAE0819) was transformed into E. coli BL21 codon plus(DE3)-RIL cells. Expression and cell harvesting were performed as described above. The pellet was resuspended in 50 mM Tris-HCl, pH 8.5. Cells were disrupted by passing through a French pressure cell. After centrifugation, the supernatant was heat-precipitated at 75 °C for 30 min and centrifuged. After buffer exchange by ultrafiltration, the supernatant was applied to a Uno-S5 column (5 ml) equilibrated with 50 mM piperazine, pH 5.3. Protein was eluted with a gradient from 0 to 1 M NaCl. Fractions containing the highest PK activity (0.3–0.4 M NaCl) were pooled and concentrated to a volume of 1 ml by ultrafiltration. The protein solution was applied to a Superdex 200 HiLoad 16/60 gel filtration column equilibrated with 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, and the eluted PK was essentially pure.

Enzyme Assays and Determination of Kinetic Parameters—The PK activity was determined up to 65 °C using a continuous assay in the direction of pyruvate formation. It was ensured that the auxiliary enzyme was not rate-limiting. One unit of enzyme activity is defined as 1 µmol of product formed per minute. The assay mixture contained for A. fulgidus and T. maritima 100 mM triethanolamine, pH 7.0, 1 mM PEP, 2 mM ADP, 5 mM MgCl2, 0.3 mM NADH, and 1 unit of LDH, and, for A. pernix and P. aerophilum 100 mM bis-Tris, pH 6.2, 1 mM PEP, 2 mM ADP, 5 mM MgCl2, 0.3 mM NADH, and 1 unit of LDH. The formation of pyruvate from 65 to 98 °C was measured by using a discontinuous assay. The standard assay mixture (250 µl) contained 100 mM Tris-Cl, 1–5 mM PEP, 2 mM ADP, 5–10 mM MgCl2. After preincubation, the reaction was started with an aliquot of PK, incubated for 15–120 s, and stopped by rapid addition of 750 µl of ice-cold buffer (100 mM Tris-HCl, pH 7.0, 0.6 mM NADH, 0.5 unit of LDH); the amount of pyruvate formed was quantified by following the oxidation of NADH at 365 nm. Kinetic parameters of PKs were determined at 65 °C using the continuous assay (see above). Six to eight different concentrations of the substrates PEP and ADP were used. The assay mixtures contained 0.3 mM NADH and 1 unit of LDH for all PKs and, specifically, as follows: A. pernix, 0.1 M bis-Tris, pH 6.2, and 0–1 mM (ADP/2 MgCl2), 0.5 mM PEP or 0–1 mM PEP, 0.5 mM ADP, 1 mM MgCl2; A. fulgidus, 0.1 M triethanolamine, pH 7, and 0–3 mM ADP/MgCl2, 0.4 mM PEP or 0–2 mM PEP, 0.2 mM ADP, 1 mM MgCl2; P. aerophilum, 0.1 M bis-Tris, pH 6.2, and 0–2.5 mM ADP/12.5 MgCl2, 1 mM PEP or 0–1 mM PEP, 1.5 mM ADP, and 7.5 mM MgCl2; and T. maritima, 0.1 M MES, pH 6.5, and 0–3 mM ADP/MgCl2, 5 mM PEP or 0–3 mM PEP, 2.5 mM ADP/MgCl2. Kinetic constants and standard errors are obtained from best-fit curves. The data points given in the figures are original measurements of one experiment; the curves drawn represent fits to a sigmoidal model or a hyperbolic model (Fig. 4, Thermotoga PK activity in the presence of AMP) according to non-linear regression analysis using the MicrocalTM OriginTM software version 5.0. In the determination of the Hill coefficients, the best-fit lines generated via linear regression analysis by the same software are shown (e.g. Fig. 1B).



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FIG. 4.
Rate dependence of pyruvate kinase from T. maritima on PEP concentration in the presence and absence of effector. No effector ({blacksquare}), 1 mM AMP (•), and 1 mM ATP ({blacktriangleup}).

 


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FIG. 1.
Rate dependence of pyruvate kinase from P. aerophilum on substrate concentrations. A, PEP saturation curve; B, Hill plot of the same data; and C, ADP saturation curve.

 

Temperature Dependence and Thermal Stability—The temperature dependence of PK activities was measured between 20 and 98 °C, using the discontinuous assay, in 100 mM triethanolamine, pH 7.0 (A. fulgidus), or 100 mM bis-Tris, pH 6.2 (A. pernix, P. aerophilum, and T. maritima) each containing 1 mM PEP, 2 mM ADP, and 5 mM MgCl2. Long term thermostability of PKs (0.5 µg in 30 µl of 100 mM triethanolamine, pH 7.0 (A. fulgidus), 1.5 µgin30 µl of 100 mM bis-Tris, pH 6.2 (A. pernix), 1.4 µg in 30 µl of 100 mM sodium phosphate buffer, pH 7.0 (P. aerophilum), and 1 µg in 30 µl of 100 mM triethanolamine, pH 7.0 (T. maritima), each at the respective temperature) were tested in sealed vials, which were incubated at temperatures between 70 and 110 °C up to 120 min. The vials were cooled for 10 min, and the remaining activity was tested in a continuous assay.

Circular Dichroism Spectroscopy—CD spectroscopy analyses were performed on a Jasco J-715 CD spectrometer. Spectra were recorded in 0.1-mm cuvettes and corrected for the signal of the solvent (20 mM sodium phosphate, pH 7.0). Secondary structure analysis and assignment to different secondary structure types were performed by the experimentally established spectra-structure correlation using the Varselec option of Dicroprot (33). Heat-induced unfolding of PKs was analyzed in temperature gradient experiments. The protein samples were dialyzed against 20 mM sodium phosphate buffer, pH 7.0, and the protein concentrations were set to 100 µg/ml. The temperature of the samples was raised at a rate of 1 °C per minute from 50 to 98 °C. Protein unfolding was followed by temperature-dependent change of a {alpha}-helical ellipticity ({phi}) at 221 nm. The observed ellipticity ({phi}obs) at a given temperature was corrected for the temperature-dependent baseline to give {phi}obs '. The fraction of unfolded protein (Xuf) was calculated using the temperature-corrected ellipticities of the folded ({phi}f') and unfolded ({phi}uf') states. Spectra were recorded before and after each temperature gradient experiment to characterize the folded and unfolded states. The fraction of unfolded protein was calculated according to the equation, Xuf = ({phi}obs ' – {phi}f')/({phi}uf' – {phi}f').

pH Dependence, Cation Specificity, and Effectors—The pH dependence of the enzymes was measured between 5.5 and 8.2 at 50 °C in the continuous assay using either bis-Tris (pH 5.5–6.5), bis-Tris-propane (pH 6.0–7.5), or Tris-HCl (pH 7.0–8.2) (A. fulgidus) and at 65 °C using either piperazine (pH 5.5–6.1), bis-Tris (pH 6.1–6.5), or triethanolamine (pH 6.5–7.5) (A. pernix, P. aerophilum, and T. maritima) (each 100 mM). Cation specificities were examined using the standard continuous assay at 50 °C (A. fulgidus) or 65 °C (A. pernix, P. aerophilum, and T. maritima) as described above by replacing MgCl2 for alternative divalent cations (Mn2+, Co2+, Ca2+, Zn2+, Ni2+, or Fe2+) at equimolar concentrations (0.1 mM, 1 mM, and 5 mM). The dependence on K+ and was tested using concentrations up to 100 mM. The following classic allosteric effectors of PKs, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, AMP, L-alanine, ribose 5-phosphate, glucose 6-phosphate, fructose 6-phosphate, citrate, and erythrose 4-phosphate (concentration range between 10 µM and5mM) were tested at 65 °C using the continuous assay as described above with both PEP and ADP concentrations near their S0.5 values: A. fulgidus, 0.4 mM MgCl2, 0.4 mM ADP, and 0.2 mM PEP; P. aerophilum, 7.5 mM MgCl2, 1.5 mM ADP, and 0.5 mM PEP; A. pernix, 1 M MgCl2, 0.5 mM ADP, and 0.3 mM PEP. In the case of A. pernix PK the effectors were preincubated with the protein at the respective temperature. The assay for T. maritima PK contained 2.5 mM ADP, 2.5 mM MgCl2, and 0.3 mM PEP in 0.1 M MES, pH 6.5 (65 °C). When effectors were tested, the substrates ADP and PEP were used at the highest purity available.

Sequence Handling—Sequence alignments were constructed with the Neighbor-joining method of ClustalX (34) using the GONNET matrix. Phylogenetic trees were constructed using both the Neighborjoining option of ClustalX as well as the Maximum-likelihood method of PROML (Phylip, version 3.6). Confidence limits were estimated by 100 bootstrapping replicates.

Sources of Organisms—A. fulgidus strain 7324 (DSM 8774), A. pernix (DSM 11879), P. aerophilum (DSM 7523), and T. maritima (DSM 3109) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyruvate Kinases from the Hyperthermophilic Archaea A. pernix, P. aerophilum, and A. fulgidus Strain 7324
In the genomes of the hyperthermophilic crenarchaeota A. pernix and P. aerophilum ORF APE0489 and PAE0819, respectively, were annotated as putative pyk genes coding for pyruvate kinase. To prove their coding function, the ORFs were cloned and functionally expressed in E. coli. The recombinant proteins were characterized. In the genome of A. fulgidus strain VC16, no pyk homologous gene was identified. Because the closely related strain A. fulgidus 7324 has been shown to contain high PK activity after growth on starch (3), PK was purified and characterized from this Archaeoglobus strain.

PK from A. fulgidus Strain 7324
Extracts of A. fulgidus grown on starch as carbon and energy source contained PK activity (0.13 unit/mg, 50 °C), which is about 5-fold higher as compared with PK activity of lactate grown cells (0.02–0.04 unit/mg) indicating a catabolic function of the enzyme during sugar degradation (3). PK was purified from starch-grown cells to homogeneity using four chromatographic steps. The enzyme was purified about 1200-fold to a specific activity of 1000 units/mg at 80 °C with a yield of 7%.

Molecular Composition and N-terminal Amino Acid Sequence
The native enzyme had an apparent molecular mass of 203 kDa and showed one 49-kDa band on SDS-PAGE indicating a homotetrameric ({alpha}4) structure of the enzyme (Table I). The N-terminal amino acid sequence (20 amino acids, aa) of the subunit was determined: MQLPSHKTKIIATIGPASRQ. An alignment of the N-terminal amino acid sequence from A. fulgidus PK with putative PKs from hyperthermophilic archaea showed the highest degree of identity with hypothetical PK from Thermococcus litoralis (18 aa identical) and Pyrococcus furiosus (15 aa identical). Surprisingly, using the N-terminal sequence of PK from A. fulgidus strain 7234, no ORF could be identified in the complete sequenced genome of closely related A. fulgidus VC 16 (35), thus confirming the absence of a pyk homologous gene in the A. fulgidus type strain.


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TABLE I
Molecular and kinetic properties of purified recombinant pyruvate kinases from A. pernix and P. aerophilum and of purified pyruvate kinase from A. fulgidus

Kinetic constants were measured at 65 °C, and standard errors are given (see "Material and Methods").

 

Functional Overexpression of ORF PAE0819 and APE0489 Encoding PKs from the Archaea P. aerophilum and A. pernix and Purification of the Recombinant PKs
ORF PAE0819 contains 1386 bp coding for a polypeptide of 461 amino acids with a calculated molecular mass of 50.3 kDa. The ORF was cloned and expressed in E. coli. The PK was purified from E. coli by heat treatment and two chromatographic steps to a specific activity of 46 units/mg at 65 °C. ORF APE0489, annotated as a putative pyk gene in A. pernix, contains 1374 bp coding for a polypeptide of 458 amino acids with a calculated molecular mass of 50.5 kDa. The ORF was cloned and expressed in E. coli. The His-tagged PK was purified by heat treatment, chromatography on Ni-NTA agarose, and a second heat treatment step at 100 °C to a specific activity of 53 units/mg at 65 °C. The purified PKs from P. aerophilum and A. pernix each showed apparent molecular masses of about 200 kDa; SDS-PAGE revealed one subunit each with apparent molecular masses of 48 and 51 kDa, respectively, indicating a homotetrameric structure of both archaeal PKs (Table I).

Catalytic Properties of PKs from Hyperthermophilic Archaea
The catalytic, thermophilic, and regulatory properties of the PKs from A. fulgidus, P. aerophilum, and A. pernix were analyzed (Table I). All archaeal PKs showed a sigmoidal saturation kinetics with respect to the substrates PEP and ADP, indicating a positive homotropic cooperative response to both substrates (Fig. 1 and Table I). PK activities of all archaea require divalent cations and were not dependent on potassium.

A. fulgidus PK—The purified enzyme had a specific activity of 1000 units/mg. The apparent S0.5 values for ADP and PEP, calculated from sigmoidal fit, were 0.4 and 0.25 mM, and the corresponding Hill coefficients were 2.1 and 1.8, respectively. PK activity required divalent cations. Rate dependence of Mg2+ showed sigmoidal kinetics, indicating cooperative response of PK to this cation. An apparent S0.5 value of 0.7 mM and a Hill coefficient of 1.3 were calculated. Alternative divalent cations were tested at concentrations of 0.1 mM, 1 mM, and 5 mM. For most cations (except Fe2+) 1 mM concentration was not inhibitory. At 1 mM concentration the highest PK activity was observed with Mg2+ (100% = 1060 units/mg at 50 °C), which could be replaced by Cu2+ (86%) and Mn2+ (63%) and less efficiently by Ni2+ (2%), Ca2+ (7%), and Zn2+ (6.5%). The pH optimum was at pH 6.6; 50% of activity was found at pH 5.5 and 7.5.

P. aerophilum PK—The specific activity of PK was 46 units/mg. The apparent S0.5 values for ADP and PEP were 1.3 mM and 0.4 mM; the calculated Hill coefficients were 2.7 and 2.8, respectively (Fig. 1). The highest activity was found with Mn2+ (100% = 200 units/mg at 65 °C) and Co2+ (80%) (each at 1 mM concentration). Remarkably, the enzyme did not show significant activity (about 1%) with Mg2+ (1 mM). The relative activity with Mg2+, however, was about 58% as compared with that with Mn2+ (100% = 0.75 unit/mg), when the cations were tested at 0.1 mM concentration. No activity was observed with Ca2+ and Zn2+. The pH optimum was at pH 6.

A. pernix PK—The specific activity of PK was 53 units/mg. The apparent S0.5 values for ADP and PEP were 0.26 and 0.1 mM, and the calculated Hill coefficients were 2.1 and 1.5, respectively. PK activity required divalent cations. Highest activities (1 mM cation) were determined with Mg2+ (100%), Co2+ (170%), and Mn2+ (160%). Ca2+ (14%), Zn2+ (11%), and Ni2+ (14%) were less efficient. With Mg2+ the enzyme showed cooperative binding and revealed a S0.5 of 0.7 mM and a Hill coefficient of 1.4. The pH optimum of the enzyme was at pH 6.1.

Temperature Optimum and Thermostability of PKs from Hyperthermophilic Archaea
A. fulgidus PK—PK activity showed a temperature optimum at 85 °C. The enzyme was highly thermostable, did not lose significant activity upon incubation at 70 °C for 120 min, and showed a half-life of about 20 min at 90 °C. At 100 °C an almost complete loss of activity was observed after 7 min. Addition of 1 M (NH4)2SO4, rather than NaCl or KCl (1 M each), effectively stabilized PK against heat inactivation at 100 °C, retaining about 50% residual activity after incubation at 120 min.

P. aerophilum PK—PK activity showed an optimum at higher than 98 °C, the highest possible temperature. The enzyme showed high stability against heat inactivation with a half-life of 220 min at 100 °C.

A. pernix PK—PK activity showed a temperature optimum higher than 95 °C (Fig. 2, A and B). The enzyme showed the highest thermostabilily of all archaeal PKs. The enzyme did not lose activity upon incubation for 120 min at 100 °C. Even at 110 °C the PK showed a half-life of about 30 min (Fig. 2C). Addition of (NH4)2SO4, NaCl, or KCl (each 1 M) did not stabilize PK activity against heat inactivation at 110 °C.



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FIG. 2.
Effect of temperature on the specific activity and thermostability of pyruvate kinase from A. pernix. A, temperature dependence of the specific activity; B, Arrhenius plot of the same data; and C, thermostability at 100 °C (•) and at 110 °C ({blacksquare}). 100% activity corresponded to 50 units/mg.

 

Thermostability of PKs from Hyperthermophilic Archaea Analyzed by CD Spectroscopy
The high stability of PKs against heat inactivation was further supported by following heat-induced unfolding of the proteins up to 98 °C by CD spectroscopy at 221 nm. Unfolding was observed only for PK of A. fulgidus showing a melting temperature (Tm) of 93 °C. No unfolding was detected with PKs from P. aerophilum and A. pernix up to temperatures of 98 °C (Fig. 3), indicating melting temperatures higher than 100 °C. This is in accordance to the higher temperature optima for catalytic activity and the thermostabilities of the latter PKs as compared with the A. fulgidus PK.



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FIG. 3.
Thermal induced unfolding of pyruvate kinases from the archaea A. fulgidus, A. pernix, and P. aerophilum and from the bacterium T. maritima measured by CD spectroscopy at 221 nm.

 

Effect of Allosteric Effectors on PKs from Hyperthermophilic Archaea
The effect of classic positive allosteric effectors for PKs of most eukarya and bacteria, such as FBP and AMP, were tested at 65 °C on PK activity from A. fulgidus, P. aerophilum, and A. pernix (see "Materials and Methods"). There was no activation effect observed by any of the ligands tested with the PKs studied. ATP has been reported to be an allosteric inhibitor of several PKs from eukarya and bacteria, inhibition being reversed by positive allosteric effectors FBP or AMP. ATP (1 mM) inhibited activities of archaeal PKs, e.g. about 55% in A. fulgidus PK (at 0.3 mM PEP, 0.2 mM ADP). Inhibition could not be reversed by the addition of FBP or AMP (1 mM each). However, ATP-induced inhibition could be reversed up to 90%, by increasing the PEP concentration from 0.3 mM to 1 mM or by the addition of 1 mM ADP, indicating competitive inhibition. Inhibition of activity by ATP, competitive to ADP and PEP, has also been described for other PKs (36).

The apparent absence of allosteric regulation by heterotropic compounds of the PKs from hyperthermophilic archaea might be due to their hyperthermophilic nature and/or due to as yet unknown different regulatory mechanism of the modified EM pathways of archaea. Thus, for comparison we characterized the PK from the hyperthermophilic bacterium T. maritima, which uses the conventional EM pathway for glucose degradation. Both the native enzyme and, for structural and functional analysis, the recombinant PK were analyzed.

Pyruvate Kinase from the Hyperthermophilic Bacterium T. maritima
Cell extracts of T. maritima grown on starch as carbon and energy source contained a 5-fold higher PK activity (0.13 unit/mg, 50 °C), as compared with PK activity of cells (0.02 unit/mg) grown on yeast extract indicating the induction of the enzyme during sugar catabolism. PK was purified from starch-grown cells to homogeneity in five chromatographic steps. The enzyme was purified about 2000-fold to a specific activity of 320 units/mg at 70 °C with a yield of 7%. The native enzyme had a molecular mass of 194 kDa and was composed of 51-kDa subunits indicating a homotetrameric structure (Table II). The N-terminal amino acid sequence of the subunit (MRSTKIVCTVGPRTD) was identical to the deduced N-terminal sequence of the ORF TM0208, which is annotated as a putative pyk gene encoding pyruvate kinase.


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TABLE II
Molecular and kinetic properties of the purified native and recombinant pyruvate kinase from T. maritima

Kinetic constants were measured at 65 °C, and standard errors are given (see "Material and Methods").

 

Functional Overexpression of TM0208 Encoding PK from T. maritima and Purification of the Enzyme
ORF TM0208 contains 1398 bp coding for a polypeptide of 466 amino acids with a calculated molecular mass of 51.9 kDa. The ORF was cloned and expressed in E. coli. The PK was purified by heat treatment in three chromatographic steps. The His-tagged PK showed a molecular mass of 210 kDa and a subunits size of 56 kDa on SDS-PAGE indicating a homotetrameric structure.

Catalytic, Thermophilic, and Regulatory Properties of Native and Recombinant PK from T. maritima
The apparent Vmax values (at 65 °C) for pyruvate formation of the native and the recombinant PK were 320 and 580 units/mg, respectively. The lower activity of the native enzyme is probably due to the damage during time-consuming purification procedure (2000-fold purification in five chromatographic steps). Both, native and recombinant PK were almost identical with respect to the following properties. The enzymes showed positively cooperative response to both ADP and PEP with apparent S0.5 values of 1.3 and 0.3 mM; the corresponding Hill coefficients were 2.9 and 2.1, respectively (Table II and Fig. 4). The pH optimum was near 6.0; 30% of the activity was found at pH 5.5 and 7.0. PK activity required divalent cations; at 1 mM concentration, Mg2+ (100%) could be efficiently replaced by Co2+ (120%) and Mn2+ (35%) rather than by Ca2+ (3%), Zn2+ (2.5%), Ni2+ (3%), or Fe2+ (1.5%). Mg2+ showed cooperative response to the enzyme with a S0.5 of1mM and a Hill coefficient of 2.3. PK activity was not dependent on monovalent cations, such as K+ and . Addition of both KCl or NH4Cl (40 mM each) resulted in a decrease of PK activity (recombinant) by 50–60%.

Temperature Optimum and Stability
PK activity (recombinant) showed an temperature optimum at 80 °C. Both native and recombinant PK showed high thermostability up to 85 °C; even at 100 °C the enzyme showed a half-life of about 20 min, but an almost complete loss of activity was observed after 120 min. Addition of (NH4)2SO4, rather than NaCl or KCl (each 1 M), stabilized PK against heat inactivation at 100 °C, retaining about 40% residual activity after 120-min incubation. Thermal unfolding of PK, as measured by CD spectroscopy, was not observed up to 98 °C (Fig. 3).

Effect of Allosteric Effectors on PK Activity
The effect of classic allosteric activators, such as AMP and FBP, and of the allosteric inhibitor ATP was tested on PK activity at 65 °C. The rate dependence of enzyme activity on increasing PEP concentrations in the presence of AMP and of ATP is shown in Fig. 4. In the absence of effectors, rate dependence of PK (recombinant) showed sigmoidal kinetics with an S0.5 value of 0.23 and a Hill coefficient of 2.2. Addition of AMP, rather than of FBP, allosterically activates the enzyme: rate dependence on PEP changed from a sigmoidal kinetics to a hyperbolic, Michaelis-Menten kinetics, paralleled by the decrease in S0.5 for PEP from 0.23 mM to a Km of 0.08 mM; the Hill coefficient decreased to 1.0, and Vmax remained almost constant. Thus, e.g. at a PEP concentration of 0.1 mM, AMP activates PK activity up to 10-fold. Conversely, the addition of ATP resulted in an allosteric inhibition of PK activity by increasing S0.5 from 0.23 to 0.5 mM; the Hill coefficient increased to 2.9, and Vmax was reduced to 70%. Inhibition by ATP was completely reversed by the addition of the activator AMP (1 mM). Thus, in contrast to the PKs from hyperthermophilic archaea, both AMP and ATP exerted their classic allosteric effects toward the hyperthermophilic PK of the bacterium T. maritima.

CD Spectra of Hyperthermophilic PKs from the Archaeon P. aerophilum and the Bacterium T. maritima
To get information about the secondary structure of PKs from hyperthermophiles, CD spectra were recorded for PK from Pyrobaculum and Thermotoga. The spectra of both PKs were almost superimposable (Fig. 5). For the PK from P. aerophilum an {alpha}-helical content of 36% and a {beta}-sheet content of 25% were estimated, which closely match the secondary structure predictions (36% {alpha}-helical and 26% {beta}-sheet) for both enzymes. The secondary structure estimations were comparable to those derived from the x-ray structures of yeast (38% {alpha}-helical and 12% {beta}-sheet) and E. coli PK (38% {alpha}-helical and 21% {beta}-sheet).



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FIG. 5.
CD spectra of pyruvate kinases from T. maritima (——) and P. aerophilum (····).

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Molecular and Thermophilic Properties—The PKs from the hyperthermophiles were characterized as homotetramers of about 200 kDa composed of 50-kDa subunits, which is a common feature of PKs from bacteria and eukarya, and of the two archaeal PKs characterized so far, from the crenarchaeon T. tenax and the euryarchaeon T. acidophilum (28, 29). In accordance with the optimal growth temperatures of the respective organism, the hyperthermophilic PKs of our study showed the highest temperature optimum and thermostability described so far. For example, PK from P. aerophilum (optimal growth temperature, 100 °C) showed a temperature optimum higher than 98 °C and was heat-resistant up to 100 °C for 2 h. In addition, thermal unfolding experiments revealed extremely high melting temperatures of the PKs near or above 100 °C. For comparison, PK from the extreme thermophilic bacterium Thermus was completely heat-inactivated in less than 10 min at 100 °C (37).

Kinetic and Regulatory Properties—All hyperthermophilic PKs require divalent cations for activity, a common property of all characterized PKs; Mn2+, Mg2+, or Co2+ being most effective. The PKs from the hyperthermophilic archaea and from Thermotoga were not dependent on monovalent cations such as K+ and . K+-independent PKs have been reported for E. coli, Corynebacterium glutamicum, and Z. mobilis and for the archaeon T. tenax (18, 22, 29, 38). PK sequences contain highly conserved K+ sites (residues 48–52 and 79–88 of the Pyrobaculum PK), including a glutamate in K+-stimulated PKs, e.g. Glu-89 of the yeast PK (Fig. 6). This glutamate is substituted in potassium-independent PKs. In accordance with the lack of the dependence on potassium, all known hyperthermophilic PKs have substituted glutamate at the equivalent position (e.g. by Arg, Lys, or Ser). The PK from the moderate thermophilic archaeon T. acidophilum, which contains this glutamate, was described to be dependent on potassium (28).



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FIG. 6.
Multiple sequence alignment of amino acid sequences of pyruvate kinases from eukarya, bacteria, and archaea. The alignment was generated with ClustalX. Conserved residues that have been proposed to be indispensable for catalytic activity as deduced from the yeast x-ray structure (26) are indicated by asterisks. The arrow indicates conserved Glu residue essential for K+ dependence. The consensus pattern is indicated by a box. The predicted secondary structure of the P. aerophilum pyruvate kinase is shown above the sequences. For accession numbers see Fig. 7.

 



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FIG. 7.
Phylogenetic relationships of pyruvate kinases from bacteria, eukarya, and archaea. The numbers at the nodes are bootstrapping values according to neighbor-joining (values on top) and maximum-likelihood (values beneath). NCBI accession numbers or SwissProt identifiers: A. pernix, BAA79454 [GenBank] ; Bac.lic., Bacillus licheniformis KPYK_BACLI; Bac.ste., Bacillus stearothermophilus S29783 [GenBank] ; Cor.glu., C. glutamicum KPYK_CORGL; Dei.rad., Deinococcus radiodurans AAF12171 [GenBank] ; E. coli1, KPY1_ECOLI; E. coli2, KPY2_ECOLI; Cat, KPY1_FELCA; Hae.inf., Haemophilus influenzae KPYK_HAEIN; Human, KPY2_HUMAN; Hyd.the., Hydrogenophilus thermoluteolus BAA95686 [GenBank] ; Lac.del., Lactobacillus delbrueckii KPYK_LACDE; Lac.lac., Lactococcus lactis B40620 [GenBank] ; Lei.mex., L. mexicana KPYK_LEIME; M. jannaschii, Methanococcus jannaschii D64313 [GenBank] ; M. acetivorans, Methanosarcina acetivorans str. C2A AAM07241 [GenBank] ; M. mazei, Methanosarcina mazei Goe1 AAM30411 [GenBank] ; Rabbit, KPY1_RABIT; P. aerophilum, AAL63053 [GenBank] ; Pyrococcus abyssi, CAB50316 [GenBank] ; P. furiosus, AAL81312 [GenBank] ; P.hor., Pyrococcus horikoshii F71171 [GenBank] ; yeast 1, KPY1_YEAST; yeast 2, KPY2_YEAST; Sal.typ.1, S. typhimurium LT2 AAL20302 [GenBank] ; Sal.typ.2, S. typhimurium LT2 AAL20804 [GenBank] ; Str.the., Streptococcus thermophilus AAF25804 [GenBank] ; Str.coe., Streptomyces coelicolor T35759 [GenBank] ; Str.coe., A3 CAB70653 [GenBank] ; Sulfolobus solfataricus, AAK41255 [GenBank] ; Sulfolobus tokodaii, BAB66695 [GenBank] ; T. acidophilum, KPYK_THEAC; Thermoplasma volcanium, BAB60191 [GenBank] ; T. tenax, AAF06820 [GenBank] ; T. maritima, AAD35300 [GenBank] ; Try.bru., T. brucei brucei KPY2_TRYBB.

 
All hyperthermophilic PKs showed a sigmoidal rate dependence for the substrates PEP and ADP and for Mg2+, indicating positively homotropic cooperative response to substrates and cations. Cooperative substrate binding has also been described for a few PKs from eukarya and bacteria. However, many PKs, including the PK of the hyperthermophilic archaeon T. tenax (29), have been reported to show hyperbolic rate dependence on ADP, suggesting a different conformational response of these PKs to ADP binding.

An important result of this study is the apparent lack of allosteric regulation of the PKs from the hyperthermophilic archaea by classic heterotropic compounds such as fructose 1,6-bisphosphate (FBP), other sugar phosphates, or AMP. Also fructose 2,6-bisphosphate, the allosteric activator of the Leishmania PK (16), did not have an effect. Thus, the hyperthermophilic archaeal PKs differ from PKs of bacteria and eukarya, which are allosterically activated by these compounds. ATP has been described to be an allosteric inhibitor of various eukaryal and bacterial PKs, with inhibitions being reversed by the activator FBP or AMP (39). In contrast, the ATP inhibition of PKs from hyperthermophilic archaea in this study is competitive to substrates PEP and ADP and could not be reversed by AMP or FBP.

An apparent absence of heterotropic allosteric regulation has also been reported for the PK of the hyperthermophilic archaeon T. tenax (29). However, the PK from the archaeon T. acidophilum has been described to be activated by AMP (28), indicating that the reduced regulatory capacity is probably not a general feature of all archaeal PKs. In contrast to the hyperthermophilic PKs, the PK from the hyperthermophilic bacterium T. maritima showed the classic allosteric response to the allosteric regulators of bacteria. It was allosterically activated by AMP and inhibited by ATP. Inhibition of ATP could be reversed by AMP.

The reasons for the absence of classic heterotropic regulation of PKs in hyperthermophilic archaea are not understood. A specific effect of high temperatures can be excluded, because the hyperthermophilic PK from Thermotoga showed the classic allosteric response. The different regulatory behaviors in hyperthermophilic archaea and Thermotoga might be due to the differences in glycolytic pathways, e.g. in archaea all EM pathways are modified, whereas in Thermotoga the classic EM pathway is operative. A comparative analysis of e.g. adenine nucleotide pools in hyperthermophilic archaea and Thermotoga might give an answer to the different response to AMP.

Sequence Alignment—PKs from the hyperthermophilic organisms T. maritima, A. pernix, and P. aerophilum show a high degree of similarity to characterized and putative PKs (37–65% similarity) of eukarya and bacteria. Thus, all PKs constitute a homologous family: besides minor deviations at variable positions, all the hyperthermophilic PKs contain the PK consensus pattern [LIVAC]-X-[LIVM](2)-[SAPCV]-K-[LIV]-E-[NKRST]-X-[DEQHS]-[GSTA]-[LIVM] (40). An alignment of these hyperthermophilic PKs with selected homologous proteins from all three domains is given in Fig. 6. Available PK crystal structures, from cat and rabbit muscle, yeast, E. coli, and L. mexicana revealed that each subunit is composed of four domains (N, A, B, and C) (eukarya) or three domains (A, B, and C) (bacteria). The domain N, located at the N terminus, is a short {alpha}-helical stretch present in eukaryotic sequences but absent in the bacterial and archaeal homologs. The domain A, which includes the catalytic site (residues 14–83 and 176–348 of the P. aerophilum PK, Fig. 6), constitutes a classic ({alpha}{beta})8 barrel structure. The domain B (residues 84–175) is a {beta}-sheet capping the catalytic domain. The domain C (residues 349–461), located at the C terminus, is an open twisted {alpha}, {beta} structure, containing the FBP binding site of the yeast enzyme. The highest degree of homology is found in the catalytic domain A. In this domain, a number of residues have been identified to be important for catalysis, according to the yeast PK structure (26). These residues are conserved in all selected PK sequences, including the PKs of this study. In contrast to the domain A, domain B and in particular C showed significantly lower sequence homology. As deduced from the structure of the yeast PK, at least eight residues (Ser-402, Thr-403, Ser-404, Thr-407, Trp-452, Arg-459, Gly-475, and His-491 of the yeast PK) have been identified in the domain C to contact the allosteric effector FBP. However, these residues are not conserved among the FBP-regulated PKs. The presence of a conserved glutamate (Glu-432) has been attributed to the non-allosteric property of the mammalian M1 isoenzyme (26). In the PKs of hyperthermophilic archaea, no glutamate at the equivalent position was found, indicating that a conserved glutamate is not a prerequisite for non-allosteric behavior of PKs. Thus, the absence of FBP regulation of the hyperthermophilic PKs cannot be explained on sequence level. So far, crystal structures of both AMP-activated PKs and PKs from hyperthermophilic archaea have not been reported. Crystallization of the AMP-regulated PK from T. maritima and of the archaeal PKs are in progress to identify the AMP binding site and to understand the structural basis for reduced regulatory properties of hyperthermophilic archaeal PKs.

Phylogenetic Affiliation—With the detailed characterization of the PKs from T. maritima, A. pernix, and P. aerophilum along with the recently available archaeal homologs, a sufficient number of PKs from all three domains of life is available allowing phylogenetic studies with respect to both evolutionary as well as functional aspects. As demonstrated in the phylogram in Fig. 7, selected PK sequences are clustered in four groups: PK bacteria I, PK archaea, PK eukarya, and PK bacteria II. The overall topology of the tree was achieved by both neighbor-joining and maximum likelihood methods and is supported by fairly good bootstrapping values. However, the lower values of some basal nodes are probably due to the influence of several factors: phylogenetic distance, regulation, physiology, evolutionary pressure, and temperature adaptation. The PK bacteria I cluster includes the majority of the bacterial PKs, and for those enzymes, which were functionally characterized, AMP has been shown to be a positive allosteric effector. Almost all archaeal PK sequences available form a separate cluster supporting the monophyletic origin of the archaeal domain. Interestingly, the PK from the hyperthermophilic bacterium T. maritima clusters within the archaeal sequences. This might reflect lateral gene transfer of the pyk gene from an hyperthermophilic archaeon into the T. maritima genome, a phenomenon that has been suggested to occur in T. maritima at high frequency (41). PKs from the archaeal cluster did not show heterotropic regulation or were regulated by AMP (Thermotoga and Thermoplasma). The PK sequence of the archaeon A. fulgidus strain 7324 is not known. N-terminal amino acid sequences indicate high identity to PKs from the archaea Pyrococcus and Thermococcus. The absence of a pyk homolog in the genome of the closely related strain, A. fulgidus VC16, might be explained by a loss of this gene; alternatively, A. fulgidus 7324 might have taken up its pyk gene via lateral gene transfer from Thermococcales. The putative PK from the Halobacterium NRC I, an outgroup with low bootstrapping support, was omitted. All eukaryotic PK sequences form a cluster. Most of them are allosterically activated by F-1, 6-BP or F-2, 6-BP (PKs from protistas). However, the absence of allosteric activation has been reported for a few eukaryal isoenzymes. The second bacterial PK cluster comprises PKs from Gram-positives with low guanine-cytosine content and {gamma}-proteobacteria species. Enzymes from this group show allosteric response to either FBP or to AMP. The two separate bacterial PK clusters might have evolved by a gene duplication in the early bacterial evolution. Alternatively, lateral gene transfers from eukaryotes to some Gram-positive and proteobacteria might be postulated. The latter hypothesis could explain the close clustering of the second bacterial group with the eukarya, as well as the allosteric regulation by FBP of isoenzymes 1 from E. coli and S. thyphimurium, a property found only in eukaryal PKs.


    FOOTNOTES
 
* This work was supported by grants of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 49-431-880-4328; Fax: 49-431-880-2194; E-mail: peter.schoenheit{at}ifam.uni-kiel.de.

1 The abbreviations used are: EM, Embden-Meyerhof pathway; GLK, glucokinases; PFK, 6-phosphofructokinases; GAP, glyceraldehyde 3-phosphate; FBP, fructose 1,6-bisphosphate; MES, 4-morpholineethanesulfonic acid; ORF, open reading frame; Ni-NTA, nickel-nitrilotriacetic acid; CD, circular dichroism; aa, amino acid(s); PEP, phosphoenolpyruvate; LDH, L-lactate dehydrogenase. Back


    ACKNOWLEDGMENTS
 
We thank Dr. J. Grötzinger (Kiel) for help in CD spectroscopic measurements, Dr. R. Schmid (Osnabrück) for N-terminal amino acid sequencing, and H. Preidel (Kiel) for mass culturing A. fulgidus 7324 and T. maritima.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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