INTRODUCTION

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As shown by several investigations, Archaea use the Embden-Meyerhof-Parnas (EMP) pathway for carbon metabolism, and in some archaeal species (the genera Pyrococcus, Thermococcus, Desulfurococcus, and Thermoproteus) the catabolic EMP pathway represents the main route for glucose degradation (37, 40). So far, in thermophilic Archaea only variants of the classical EMP pathway have been found. Common to these variants is the irreversible oxidation step transforming glyceraldehyde-3-phosphate directly to 3-phosphoglycerate by a ferredoxin-dependent glyceraldehyde-3-phosphate oxidoreductase (GAP:FdOR) found in Pyrococcus, Thermococcus, and Desulfurococcus strains (37, 43) or by a nonphosphorylating NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN) found in Thermoproteus tenax (4, 15, 38). These enzymes substitute for the combined action of conventional phosphorylatingbut reversibleglyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase, which, however, allows ATP formation.

We chose T. tenax, a hyperthermophilic, facultatively heterotrophic member of the kingdom Crenarchaeota, as an object for regulatory studies on archaeal carbohydrate metabolism. T. tenax is able to grow chemolithoautotrophically on CO₂, H₂ and S⁰, as well as chemoorganoheterotrophically in the presence of S⁰ and carbohydrates such as glucose, starch, and amylose (45). The variant of the EMP pathway in T. tenax is characterized by a bidirectionally working, nonallosteric pyrophosphate-dependent 6-phosphofructokinase (PP_i-PFK) and by two GAPDHs differing in their cosubstrate specificity (15, 39). Whereas the phosphorylating NADP⁺-dependent GAPDH seems to fulfill a role in gluconeogenesis (N. A. Brunner, B. Siebers, and R. Hensel, manuscript in preparation), functional analyses of the NAD⁺-dependent GAPDH (GAPN) of T. tenax revealed that this enzyme is involved in glycolysis and represents a central control point of the pathway (4). With its regulatory properties (allosteric inhibition by NADP(H), NAD(H), and ATP; allosteric activation by AMP, ADP, glucose-1-phosphate, and fructose-6-phosphate), the enzyme seems to compensate for the missing regulatory potential of PFK. To investigate whether the integration of an irreversible, highly regulated GAPN in the catabolic EMP pathway of T. tenax impacts the functional phenotype of PK, usually the second site of glycolytic regulation, we focused on the enzymic properties of PK and its expression in response to growth conditions.

In Bacteria and Eucarya, the enzyme is functionally and structurally well characterized. In all Eucarya spp. investigated so far and some bacteria, the activity of PK depends on the presence of fructose-1,6-bisphosphate or other sugar phosphates, whereas PKs of the majority of bacteria and plastids show basal activity without effectors and are mostly regulated by adenosine phosphates (i.e., by the energy charge of the cell). More than 50 primary structures of bacterial and eucaryal enzymes are available in databases. Three-dimensional structures have been determined for one bacterial and three eucaryal enzymes (18, 24, 26, 29). Recently, the mode of ATP binding (23) and allosteric activation of PK by FBP were described at the atomic level (18). The enzyme is a homotetramer in almost all organisms, although dimeric PKs have been reported for Schizosaccharomyces pombe (30, 31) and Zymomonas mobilis (32). Each subunit folds into four distinct domains (designated N, A, B, and C). The first domain, N, is characterized by a short -helix at the N terminus, which, however, is absent in some bacterial PKs, such as the FBP-activated enzyme of Escherichia coli (26). The catalytical A domain folds into the common symmetrical /₈-barrel, sharing this topology with two other glycolytic enzymes: triosephosphate isomerase and aldolase. The third domain, B, consists of a small -barrel forming a cap over the active site. Finally, domain C located at the C terminus exhibits an / open-sheet motif and usually displays regulatory functions. In contrast to bacterial and eucaryal enzymes, little is known about the structure and function of archaeal PKs. Up to now only the enzymic properties of the PK of Thermoplasma acidophilum could be determined (34). The enzyme represents a homomeric tetramer with a molecular mass of 250 kDa and displays cooperative substrate binding; its substrate affinity is increased by AMP. Genome sequencing projects published so far have revealed the presence of PK coding genes (pyk) in Pyrobaculum aerophilum (11), Methanococcus jannaschii (5), and Pyrococcus horikoshii (19), but no information on the enzymic properties of these enzymes is available. By analyzing T. tenax PK, we wanted to better understand the control mechanisms governing the central carbon metabolism of that organism and to get better insights into the structure and function of the archaeal PK in general.

	MATERIALS AND METHODS

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Chemicals and plasmids. Phosphoenolpyruvate (PEP) and rabbit muscle lactate dehydrogenase (LDH) were from Sigma, Munich, Germany. ADP, ATP, NAD⁺-NADH, and NADP⁺-NADPH were purchased from Gerbu, Gaiberg, Germany. All other chemicals (Pro Analysis grade) were from Sigma, Fluka, or Merck. Cloning of genomic DNA was performed with plasmids pUC19 (MBI Fermentas) and pBluescript IIKS(+) (Stratagene). For heterologous expression, the vector pJF118EH (13) was used.

Bacterial strains and growth conditions. Mass cultures of T. tenax Kra 1 (DSM 2078) were grown as described previously (38). For cloning and expression experiments, E. coli DH5 (Life Technologies) was grown as described earlier (36).

Enzyme assay. The standard assay was performed at 50 or 60°C with 100 mM Tris-HCl (pH 7.0) at the respective temperature, 0.5 mM NADH, 4 U of LDH (rabbit muscle; Sigma), 20 mM PEP, 5 mM ADP, and 10 mM MgCl₂. Enzyme activity was measured by monitoring the decrease in absorption at 366 nm. Reactions were started by adding the heat-labile substrate PEP (Fig. 1). Enzyme concentrations ranged from 2 to 10 µg of protein/ml. The enzyme reaction was monitored for the first 1 to 2 min.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Arrhenius plot of PEP decay. A solution containing 50 mM HEPES (pH 7.0)-10 mM PEP was incubated at various temperatures, and the residual PEP concentration was determined by the enzyme assay described in the text.

Purification of PK from T. tenax. Preparation of crude extracts from T. tenax, heat treatment, ion-exchange chromatography, and hydrophobic interaction chromatography were performed as described previously (40). Fractions containing PK activity were pooled, dialyzed against 50 mM HEPES-KOH (pH 7.0) containing 10 mM 2-mercaptoethanol and 300 mM KCl, concentrated by membrane filtration (Centricon 30; Amicon), and subjected to gel filtration on a HiLoad 26/60 Superdex column (Pharmacia; 60 by 2.6 cm; flow rate, 0.7 ml/min) equilibrated in the same buffer. Pooled fractions containing PK activity were dialyzed and applied to a AMP-Sepharose column (Sigma; 6 by 1 cm; flow rate, 0.2 ml/min) equilibrated with 50 mM HEPES (pH 7.0) and 10 mM 2-mercaptoethanol. Homogeneous protein was eluted by equilibration buffer containing 100 mM KCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described earlier (22). Determination of protein concentration was done by using the DC Protein Assay (Bio-Rad).

Molecular mass determination. The native molecular mass was determined by gel filtration on a HiLoad 26/60 Superdex 200 preparatory-grade column (Pharmacia) as described elsewhere (40). The molecular mass of subunits was determined by denaturating SDS-PAGE with SDS-7 Marker (Sigma) as the standard.

CNBr fragmentation and protein sequencing. Because the enzyme turned out to be N-terminally blocked, the protein was cleaved with CNBr (27). Resulting peptides were separated by isoelectric focusing in the first dimension and by tricine-SDS-PAGE in the second dimension as described previously (14). Peptides and proteins were immobilized on ProBlott membranes (Applied Biosystems) by semidry electrotransfer (10). Sequencing was performed by automated Edman degradation with a gas-phase Sequenator 473A (Applied Biosystems). The partial sequence of the following two peptides were determined: peptide 1, RPLQITAGARVSFKLAEKGDGFVPVPRREFF; and peptide 2, LDGKLVLRIISAAQ.

Cloning and sequencing of the coding gene. Genomic DNA was prepared as described earlier (44), as modified by Meakin et al. (28). The gene encoding PK was identified by hybridization with a degenerated oligonucleotide 5'-TTAGATGGIAARYTNGT3' deduced from the hexapeptide LDGKLV. For hybridization, the oligonucleotide was labeled with digoxigenin according to the manufacturer's instructions (Roche Diagnostics). DNA was transferred to nylon membranes (Nytran; Schleicher & Schuell) by capillary blotting (6). Southern blots were hybridized at room temperature in 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and washed two times at 37°C in 1× SSC. A strongly hybridizing 3-kb BamHI/HindIII-fragment was selected, cloned, and sequenced with the aid of an Automated Laser Fluorescence DNA Sequencer (Amersham Pharmacia).

Expression of the pyk gene from T. tenax in E. coli. For expression of the pyk gene, the coding region was cloned into pJF118EH via two new restriction sites (EcoRI and BamHI) created with the mutagenic primers 5'-GATCGCGCGAGTAGAATTCATGTTCAC-3' and 5'-GTTTACTCAGGGATCCGTAATTTAATAG-3'. The sequence of the clone expressing the pyk gene was confirmed by sequencing of both strands. Expression in E. coli DH5 cells was performed by standard procedures (36).

Purification of T. tenax PK from transformed E. coli. First, 5 g of E. coli cells was resuspended in 10 mM potassium phosphate buffer (pH 7.3) containing 30 mM 2-mercaptoethanol and passed three times through a French press cell at 150 MPa. Then, after centrifugation (20,000 × g for 30 min), the supernatant was heat precipitated (80°C for 30 min) and centrifuged again. Homogeneous enzyme preparations were obtained by chromatography on AMP-Sepharose as outlined above for the enzyme prepared from T. tenax.

Northern blot analyses. Total RNA was prepared from autotrophically and heterotrophically grown cells by using TRIzol reagent according to the manufacturer's instructions (Life Technologies, Eggenstein, Germany). The integrity of the RNA was routinely checked by agarose gel electrophoresis by using a morpholine propane sulfonic acid (MOPS)-6% formaldehyde buffer system, and the concentration was determined spectrophotometrically at  = 260 nm. Digoxigenin-labeled antisense mRNA was obtained by in vitro transcription from the T7 promoter of vector pSPT 19 (Roche Diagnostics). For that purpose, the coding region of the T. tenax pyk gene was inserted into the EcoRI/BamHI restriction sites of the vector. Hybridization was carried out at 68°C overnight in DIG Easy Hyb solution (Roche Diagnostics), and stringency washes were performed in 0.1× SSC-0.1% SDS at 68°C. Densitometric analyses were performed by using a laser densitometer Ultroscan XL (Pharmacia LKB) according to the manufacturer's instructions.

Primer extension analyses. Primer extension was performed by using a protocol of Kuo et al. (21), with the modification that the hybridization buffer contained 0.3 M KCl, 2 mM EDTA, and 20 mM Tris-HCl buffer (pH 8.3). RNA from heterotrophically as well as from autotrophically grown cells of T. tenax was used as a source for cDNA synthesis. To map the pyk transcription start site, the 5'-³²P-labeled antisense oligonucleotide PK1PEX (5'-CCTCCGATGGCGATGCGT-3'), which is complementary to positions 103 to 120 of the pyk gene, was used as primer for cDNA synthesis and accompanying sequencing reactions. The labeled cDNA products were analyzed on a denaturating polyacrylamide gel (5%), along with a sequence ladder generated with the same oligonucleotide as the primer.

Sequence handling and phylogenetic analyses. Homology searches were performed with BLASTN at The National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/BLAST). Sequences were extracted from GenBank or from the NCBI, i.e., the Microbial Genome Database (http://www.ncbi.nlm.nih.gov/BLAST/unfinishedgenome.html), and first aligned with CLUSTAL W (42). This alignment was refined manually by using the MUST program package (33). Regions of uncertain alignment were omitted, leaving 462 amino acid positions for analysis. For the phylogenetic tree the topology of the Maximum Parsimony Bootstrap analysis was used, obtained by PAUP version 4.0d65 with 500 bootstrap replicates and four-times-random addition (41). Maximum likelihood analyses were performed by using MOLPHY version 2.3 (1), starting with the NJDIST tree, using the local rearrangement option (data not shown). Distance analysis were performed with the MUST package using the Kimura correction and the neighbor-joining method (35), including 1,000 bootstrap replicates. The T. tenax pyk gene sequence is deposited at GenBank (AF065890).

	RESULTS

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Specific activity of PK in autotrophically and heterotrophically grown T. tenax cells, purification of the enzyme, and molecular mass determination. As revealed from measurements with dialyzed crude extracts, cells grown in the presence of glucose showed a specific activity of PK approximately four times higher (0.17 U/mg) than cells grown with CO₂ as the only carbon source (0.04 U/mg of protein), thus supporting its catabolic function. From 15 g (wet weight) of heterotrophically grown cells, 0.2 mg of homogeneous protein was recovered with a specific activity of 45 U/mg, a value corresponding to a recovery of 12% (Table 1). The enzyme migrated as a single band on SDS-PAGE with an apparent molecular mass of 48 kDa (Fig. 2). Molecular mass determination under nondenaturating conditions yielded values of 200 + 10 kDa, indicating that the PK of T. tenax is a homomeric tetramer like the majority of PKs known.

View larger version (81K): [in this window] [in a new window]   FIG. 2.   Purification of PK from T. tenax and of recombinant PK. (M, marker; RE, crude extract; HF, heat precipitation; QS, anion-exchange chromatography; PS, hydrophobic interaction chromatography; GF, gel filtration; AMP, AMP-Sepharose).

Nucleotide sequence of the pyk gene and its flanking regions. Southern hybridizations with a degenerated oligonucleotide probe derived from the N-terminal sequence of a CNBr-fragment of the protein gave a strong positive signal with a 3-kb BamHI/HindIII fragment of the genomic DNA of T. tenax. Subsequent sequencing revealed two open reading frames (see GenBank accession no. AF065890). One of them was identified as a pyk gene by correspondence of the deduced amino acid sequence with the partial peptide sequence and by sequence similarity with known PKs from Bacteria and Eucarya. The second open reading frame starting 116 bp downstream of the pyk gene (i.e., the same direction as pyk) showed 35 to 50% identity to cysteinyl-tRNA synthetases from other organisms of the three domains of life.

Deduced amino acid sequence of PK. Comprising only 446 residues, the T. tenax enzyme represents one of the shortest PK sequences known. Like the protein sequences deduced from the putative pyk genes of the Archaea P. horikoshii and M. jannaschii, the PK sequence of T. tenax is about 10% shorter than those of Bacteria and Eucarya. In an alignment with 52 representatives of the three domains of life, the sequence of the T. tenax PK showed overall amino acid identities ranging from 27 to 42%. The comparison revealed that the catalytically essential residues (ARGDL) at positions 236 to 240 and residues involved in subunit binding (PTRAE) at positions 283 to 287 (Fig. 3; assigned on the basis of three-dimensional analyses of the PK from yeast [18]) are conserved in the T. tenax enzyme. These conservative residues are located in the domains A and B. Surprisingly, no preferred similarity to the translated sequences of the putative pyk genes of the Archaea P. aerophilum, M. jannaschii, and P. horikoshii were observed: the archaeal sequences neither shared specific sequence signatures nor showed significantly higher identity values with each other (28.4 to 36.2% identity) than with the homologues of Bacteria or Eucarya.

View larger version (13K): [in this window] [in a new window]   FIG. 3.   Partial sequence alignment of PK from T. tenax with PKs from eucaryal, bacterial, and archaeal representatives. GenBank entries: YEAST, S. cerevisiae (U12980); CAEEL, C. elegans (Z69385); ECOLI1, E. coli PK1 (AE000262); ECOLI2, E. coli; PK2 (AE000279); METJA, M. jannaschii (U67468). THETX, T. tenax (the present study). Conserved residues are marked with an asterisk. Residues in boldface type indicate potassium binding sites (A) or subunit and metal binding sites (B), respectively. The secondary structure elements above the sequences are deduced from the three-dimensional structure of yeast enzyme (18).

Transcript analyses. Northern blot analyses with total RNA from autotrophically and heterotrophically grown cells using antisense pyk mRNA as a probe resulted in a strong hybridization signal with a 1.3-kb andto a significantly lesser extentwith a 1.6-kb transcript (Fig. 4A). As revealed by densitometric analyses, the 1.3-kb transcript gave a signal approximately four times stronger with heterotrophic cells compared to autotrophic cells, thus corresponding well with the fourfold-higher PK activity in heterotrophically grown cells. In contrast, the signal of the 1.6-kb transcript remained at a rather low level under both growth conditions (Fig. 4A). Whether this significantly weaker signal can be assigned to a transcript initiated from an alternative promoter of the pyk gene is currently being analyzed.

View larger version (45K): [in this window] [in a new window]   FIG. 4.   Northern blot analysis of pyk mRNA and determination of the transcript start site. (A) Northern blot analysis of pyk transcripts with RNA from autotrophically (lane 1) and heterotrophically (lane 2) grown cells probed with pyk antisense mRNA. (B) Mapping of the pyk transcription start by primer extension. The transcript begins at position 1 with regard to the start codon of the pyk gene (marked by an asterisk). The primer extension products from autotrophically grown cells (lane 1) and heterotrophically grown cells (lane 2), together with the sequence ladder (lanes A, C, G, and T), are shown. (C) Nucleotide sequence of the sense strand of the pyk region. The position of a putative box A promoter element (marked in boldface) and the starting point of transcription (marked by an arrow) are indicated.

Heterologous expression of the pyk gene. For functional and structural investigations, the pyk gene was expressed in E. coli. About 1 mg of homogeneous enzyme could be obtained from 1 g (wet weight) of recombinant E. coli DH5 cells. As shown in Fig. 2 and Table 2, the enzyme isolated from T. tenax and from transformed E. coli exhibited virtually the same properties with respect to molecular mass and the kinetic parameters for substrate, cosubstrate, and Mg²⁺ saturation, indicating a highly similar phenotype for both enzymes. Therefore, the screening experiments for ligands influencing the enzymic properties were performed with the better available recombinant enzyme.

Enzymic properties. The enzyme assays were conducted at only 50°C because of the heat-labile mesophilic auxiliary enzyme used in the coupled optical test. Like known PKs, the enzyme of T. tenax showed an absolute requirement for divalent cations. However, of the metal ions tested (Mg²⁺, Mn²⁺, Co²⁺, Fe²⁺, Ni²⁺, and Cd²⁺), activity could only be observed with Mg²⁺, Mn²⁺, and Co²⁺. The highest V_max values were obtained with Mn²⁺ and Mg²⁺ (Table 2). Co²⁺ allowed only one-tenth of the maximal activity.

View larger version (9K): [in this window] [in a new window]   FIG. 5.   PEP saturation of the PK of T. tenax in the presence of 20 mM MgCl2.

Phylogenetic analyses. Phylogenetic trees were constructed based on the deduced amino acid sequences of 52 pyk genes from Bacteria, Eucarya, and Archaea by using maximum parsimony, distance matrix, and maximum likelihood (ML) methods. The tree in Fig. 6 was constructed based on the topology of the PAUP bootstrap consensus tree using the user-defined tree option of the MOLPHY package. All three methods resulted in the same dichotomic tree structure, dividing the PK enzymes into two discrete clusters. PK cluster I contains all sequences of the eucaryal cytoplasm, all sequences of low-GC gram-positive bacteria, and some of the gamma proteobacteria, whereas PK cluster II includes members of most bacterial phyla (except of low-GC gram-positive bacteria), plastids, and Archaea.

View larger version (47K): [in this window] [in a new window]   FIG. 6.   Phylogenetic tree of PK. The tree was constructed by using the user-defined tree option of the MOLPHY package (1), based on the topology of the PAUP* Bootstrap Consensus tree (41). The numbers at certain nodes are bootstrap proportions; upper values correspond to the PAUP* Bootstrap analysis (500 replicates, four-times-random addition), and lower values correspond to the Kimura distance neighbor-joining analysis (1,000 replicates). Only values of >40% are shown in the figure. The sequence of the T. tenax PK is shown in boldface. The accession numbers for all PK sequences used for the phylogenetic analysis that were found in GenBank, in addition to sequences that were identified by BLAST search and downloaded at the NCBI, were as follows: Xenopus sp. (U03878), Gallus gallus (J00903), Caenorhabditis elegans 1 (Z69385), Caenorhabditis elegans 2 (Z81068), Drosophila melanogaster (AF06247), Saccharomyces cerevisiae 1 (U12980), Saccharomyces cerevisiae 2 (Z75255), Trichoderma reesei (L07060), Aspergillus niger (S38698), Schizosaccharomyces pombe (Z69380.1), Trypanoplasma borrelii (X77255), Leishmania mexicana (X74944), Trypanosoma brucei (X57951), Solanum tuberosum (cytosol, JC1481), Glycine max (cytosol, L08632), Arabidopsis thaliana (cytosol, AL022223), Nicotiana tabacum (cytosol, Z29492), Escherichia coli type I (AE000262), Salmonella typhimurium (X99945), Bacillus stearothermophilus (D13095), Bacillus psychrophilus (D31954), Lactobacillus delbrueckii (X71403), Ricinus communis 1 (chloroplast, M64737), Nicotiana tabacum 1 (chloroplast, Z28374), Ricinus communis 2 (chloroplast, M64736), Nicotiana tabacum 2 (chloroplast, Z28373), Borrelia burgdorferi (AE001141), Mycoplasma pneumoniae (AE000052), Mycoplasma genitalium (U39698), Chlamydia trachomatis (AE001306), Methanococcus jannaschii (U67468), Pyrococcus horikoshii (AP000002), Haemophilus influenzae (U32831), Escherichia coli type II (AE000279), Agrobacterium vitis 1 (U32375), Agrobacterium vitis 2 (U25634), Methylobacterium extorquens (U87316), Zymomonas mobilis (AF079586), Mycobacterium intracellulare (U65430), Mycobacterium tuberculosis (Z95554), Corynebacterium glutamicum (L27126), Synechocystis sp. strain 1 (D90907), and Synechocystis sp. strain 2 (D64004).

	DISCUSSION

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Functional and structural properties of PK of T. tenax and its coding gene. With the T. tenax enzyme, an archaeal PK could be assigned for the first time to its coding gene. Common to known PKs, the T. tenax enzyme is N-terminally blocked. As shown, however, by comparing the enzymic properties of the PK purified from T. tenax with those of the recombinant enzyme, the putative N-terminal modification does not influence the functional properties of PK.

Phylogeny. One of the most striking features of the phylogenetic tree based on PK sequences is its dichotomic topology. Interestingly, this topology coincides with the differentiation of PK enzymes into two basic phenotypes. Thus, as known so far, all PK cluster I enzymes are active only in the presence of fructose-1,6-bisphosphate and/or other sugar phosphates, whereas the PK cluster II enzymes possess basal activity also without effectors but are mostly regulated by AMP and ATP, i.e., by the energy charge of the cell. The PK of T. tenax fits very well into this framework: like other PK II enzymes, the enzyme does not need heterotropic effectors for activity.

Physiological role. The PKs of Bacteria and Eucarya represent the second control point of the catabolic EMP pathway adjusting the level of glycolytic intermediates for degradation or biosynthetic purposes and, by controlling the flux through the pathway, also regulating the level of ATP and GTP in the cell. To adapt immediately to changing intracellular and extracellular conditions, the PK activity is, like the ATP-dependent PFK, controlled at the protein level, mainly by allosteric mechanisms, whereas long-term adaptation is achieved on the gene level. As a typical catabolic enzyme, the expression of its coding gene is significantly reduced under anabolic conditions to avoid futile cycling of PEP (8).