From the Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 19, Kiel D-24118, Germany
Received for publication, October 8, 2002 , and in revised form, March 21, 2003.
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ABSTRACT |
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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Purification of PK from A. fulgidusThe 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. maritimaThe 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 (0.150.3 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 EnzymeORF 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--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 EnzymeORF 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 EnzymeORF 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 (0.30.4 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 ParametersThe 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, 15 mM PEP, 2 mM ADP, 510 mM MgCl2. After preincubation, the reaction was started with an aliquot of PK, incubated for 15120 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 01 mM (ADP/2 MgCl2), 0.5 mM PEP or 01 mM PEP, 0.5 mM ADP, 1 mM MgCl2; A. fulgidus, 0.1 M triethanolamine, pH 7, and 03 mM ADP/MgCl2, 0.4 mM PEP or 02 mM PEP, 0.2 mM ADP, 1 mM MgCl2; P. aerophilum, 0.1 M bis-Tris, pH 6.2, and 02.5 mM ADP/12.5 MgCl2, 1 mM PEP or 01 mM PEP, 1.5 mM ADP, and 7.5 mM MgCl2; and T. maritima, 0.1 M MES, pH 6.5, and 03 mM ADP/MgCl2, 5 mM PEP or 03 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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Temperature Dependence and Thermal StabilityThe 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 SpectroscopyCD 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
-helical ellipticity (
) at 221 nm. The observed ellipticity
(
obs) at a given temperature was corrected for the
temperature-dependent baseline to give
obs '. The
fraction of unfolded protein (Xuf) was calculated using
the temperature-corrected ellipticities of the folded
(
f') and unfolded (
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 =
(
obs '
f')/(
uf'
f').
pH Dependence, Cation Specificity, and EffectorsThe 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 5.56.5), bis-Tris-propane
(pH 6.07.5), or Tris-HCl (pH 7.08.2) (A. fulgidus) and
at 65 °C using either piperazine (pH 5.56.1), bis-Tris (pH
6.16.5), or triethanolamine (pH 6.57.5) (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 HandlingSequence 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 OrganismsA. 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).
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RESULTS |
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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 (0.020.04
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 (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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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 PKThe 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 PKThe 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 PKThe 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 PKPK 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 PKPK 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 PKPK 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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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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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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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 5060%.
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 -helical content of 36% and a
-sheet
content of 25% were estimated, which closely match the secondary structure
predictions (36%
-helical and 26%
-sheet) for both enzymes. The
secondary structure estimations were comparable to those derived from the
x-ray structures of yeast (38%
-helical and 12%
-sheet) and
E. coli PK (38%
-helical and 21%
-sheet).
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DISCUSSION |
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Kinetic and Regulatory PropertiesAll 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 4852 and 7988 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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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 AlignmentPKs from the hyperthermophilic organisms
T. maritima, A. pernix, and P. aerophilum show a high degree
of similarity to characterized and putative PKs (3765% 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 -helical stretch
present in eukaryotic sequences but absent in the bacterial and archaeal
homologs. The domain A, which includes the catalytic site (residues
1483 and 176348 of the P. aerophilum PK,
Fig. 6), constitutes a classic
(
)8 barrel structure. The domain B (residues
84175) is a
-sheet capping the catalytic domain. The domain C
(residues 349461), located at the C terminus, is an open twisted
,
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 AffiliationWith 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 -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.
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FOOTNOTES |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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