Journal of Bacteriology, June 2002, p . 3401-3405, Vol . 184, No . 12

Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

Corné H . Verhees,^1* Jasper Akerboom,¹ Emile Schiltz,² Willem M . de Vos,¹ and John van der Oost¹

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands,¹ University of Freiburg, Freiburg, Germany²

Received 12 December 2001/ Accepted 15 March 2002

FBPase is an essential regulatory enzyme in the gluconeogenic pathway . It converts D-fructose-1,6-bisphosphate to D-fructose-6-phosphate, an important precursor in biosynthetic pathways . Generally, a divalent metal ion such as Mg²⁺, Mn²⁺, Co²⁺, or Zn²⁺ is required for catalytic activity . The three-dimensional structures of several FBPases have been elucidated (9, 24, 26), and all exhibit a typical sugar phosphatase fold (http://scop.mrc-lmb.cam.ac.uk/scop) .

It has recently been reported that the inositol monophosphatase (I-1-Pase) (EC 3.1.3.25) from Methanococcus jannaschii (MJ0109) exhibits FBPase activity, and it has been suggested that this enzyme might be the missing FBPase in archaea (20) . In addition, MJ0109 orthologs in Archaeoglobus fulgidus and Thermotoga maritima show FBPase activity (20) . In an attempt to complete the set of determined glycolytic and gluconeogenic enzymes in P . furiosus, we cloned and expressed the MJ0109 ortholog from P . furiosus (48% identity on the amino acid level) in Escherichia coli and investigated its ability to function as a thermoactive FBPase .

Transcript analysis and cloning of fbpA. An ortholog (fbpA) of MJ0109 (2) was identified in the P . furiosus genome database (http://www.genome.utah.edu) . This ortholog was originally designated as an extragenic suppressor, suhB . The start of the fbpA gene was predicted based on the presence and proper spacing of a potential Shine-Dalgarno sequence and on multiple alignment of the deduced amino acid sequence with those of related enzymes (Fig . 1) . To test whether the fbpA gene was transcribed in P . furiosus, total RNA was isolated from a pyruvate-grown P . furiosus culture (40 mM) as described previously (25) . The presence of the fbpA transcript was confirmed (data not shown) by using a reverse transcription-PCR system according to the instructions of the manufacturer (Promega) with 1 µg of P . furiosus RNA and the primers BG977 and BG978 (see below) . Moreover, a recent genome-based microarray analysis of P . furiosus also revealed the expression of fbpA (designated suhB) (18) .

 
The fbpA gene (765 bp) was PCR amplified from chromosomal DNA of P . furiosus as described previously (21) with the primers BG977 (5'-GCGCGTCATGAAGCTTAAGTTCTGGAGGG [with the BspHI restriction site underlined], sense) and BG978 (5'-GCGCGGATCCCTACTCCAGTAAGCTTAAAATTGTTTT [with the BamHI restriction site underlined], antisense) . The PCR product was digested with BspHI/BamHI and cloned into E . coli XL1-Blue by use of an NcoI/BamHI-digested pET24d vector according to established procedures and with 50 µg of kanamycin/ml for selection . Subsequently, the resulting plasmid, pLUW558, was transformed into E . coli BL21(DE3) .

Overexpression and purification of FBPase. An overnight culture of E . coli BL21(DE3) harboring pLUW558 was used as a 1% inoculum in 0.5 liter of Luria-Bertani medium with 50 µg of kanamycin/ml . Gene expression was induced by adding 0.1 mM isopropyl-1-thio-ß-D-galactopyranoside (IPTG) at an optical density at 600 nm of 0.5 . Growth was allowed to continue for 10 h at 37°C, and cells were harvested by centrifugation (2,200 x g for 20 min at 4°C) and resuspended in 10 ml of 50 mM Tris-HCl buffer, pH 8.0 . The cells were disrupted by French press treatment (100 MPa), and cell debris was removed by centrifugation (10,000 x g for 20 min at 4°C) . The resulting cell extract was heat treated for 30 min at 80°C, and the precipitated proteins were removed by centrifugation (10,000 x g for 30 min at 4°C) . The heat-stable cell extract was filtered through a 0.45-µm-pore-size filter and applied to a MonoQ HR 5/5 column (1 ml; Amersham Pharmacia Biotech) equilibrated with 50 mM Tris-HCl buffer, pH 8.0 . The FBPase activity eluted at 0.37 M NaCl in a linear gradient of 0.0 to 1.0 M NaCl . Active fractions were pooled and concentrated 20-fold to a final volume of 100 µl by use of a filter with a 10-kDa cutoff (Microsep; Pall Filtron) . The concentrated pool was loaded on a Superdex 200 HR 10/30 gel filtration column (24 ml; Amersham Pharmacia Biotech) equilibrated with 50 mM Tris-HCl buffer, pH 7.8, containing 100 mM NaCl . The elution pattern (not shown) suggested that the active configuration was a dimer (66.8 kDa) of two identical 33-kDa subunits, which is in good agreement with the results of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (data not shown) . The calculated size of the subunit was slightly smaller, i.e., 27.9 kDa . The purified enzyme was desalted in 50 mM Tris-HCl buffer, pH 8.0, by use of a filter with a 10-kDa cutoff (Microsep; Pall Filtron) . From 2.7 g of cell paste consisting of E . coli BL21(DE3) containing pLUW558, a total of 27.7 mg of FBPase was purified to 95% purity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (data not shown) . To ensure that the detected activity corresponded to the P . furiosus FBPase, the N-terminal sequence of the purified enzyme was determined by the Edman degradation method to be Met-Lys-Leu-Lys-Phe-Trp-Arg-Glu-Val-Ala-Ile-Asp-Ile-Ile-Ser-Asp-Phe-Glu-Thr-Thr-Ile-Met-Pro-Phe, revealing that the obtained amino acid sequence exactly matched the N-terminal sequence of the translated fbpA from P . furiosus (Fig . 1) . This indicates that the P . furiosus FBPase had been produced and purified successfully .

Temperature dependence of FBPase. For the determination of the temperature optimum, an appropriate amount of purified FBPase (6 to 30 ng) was incubated in 1-ml crimp-sealed vials containing 100 mM MOPS (morpholinepropanesulfonic acid) buffer, pH 7.4, and 10 mM MgCl₂ . The vials were submerged in an oil bath at temperatures varying from 20 to 120°C and preheated for 2 min, and the enzyme reaction was initiated by the injection of 15 mM fructose-1,6-bisphosphate . At different times up to 15 min, the reaction was stopped by transferring the vials to a mixture of ice and ethanol . Aliquots were taken, and the amount of fructose-6-phosphate formed was determined spectrophotometrically by measuring the reduction of NADP⁺ (340 nm) at room temperature in an assay with glucose-6-phosphate isomerase (EC 5.3.1.9) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49), both from Saccharomyces cerevisiae . A linear increase in fructose-6-phosphate production over time was observed, indicating that no P . furiosus FBPase was inactivated during incubation . The P . furiosus FBPase showed maximal activity at approximately 100°C (data not shown) .

In accordance with first-order inactivation kinetics (data not shown), the enzyme (18 µg/ml) lost 50% of its activity after incubation for 2 h at 100°C in 50 mM Tris-HCl buffer, pH 8.0 . For the determination of the melting temperature, P . furiosus FBPase was dialyzed extensively against a 100 mM sodium phosphate buffer, pH 8.0, and diluted to 0.3 mg/ml in dialysis buffer . After being degassed for 10 min, the samples were analyzed against the dialysis buffer in a differential scanning microcalorimeter (VP-DSC; MicroCal) between 50 and 125°C at 0.5°C/min . Enzyme scans were corrected with a buffer-buffer baseline, and data were analyzed with the MicroCal Origin 5.0 SR2 software package . For FBPase, an apparent melting temperature of 107.5°C was determined (data not shown), which is in good agreement with the inactivation kinetics .

Catalytic properties. The kinetic parameters of the P . furiosus FBPase were determined discontinuously at 85°C by varying the concentration of fructose-1,6-bisphosphate (between 0.005 and 5 mM) and by measuring the release of inorganic phosphate at room temperature as described previously (8) . The 0.2-ml assay mixture contained 50 mM Tris-HCl buffer (pH 8.0; room temperature), 10 mM MgCl₂, and 0.4 µg of purified FBPase . At this temperature, the K_m and V_max of the P . furiosus FBPase with fructose-1,6-bisphosphate were 0.32 ± 0.03 mM and 12.2 ± 0.1 U/mg, respectively, resulting in a catalytic efficiency (k_cat/K_m) of 17.7 s^-1 mM^-1 . The determined affinity of the purified FBPase for fructose-1,6-bisphosphate is in good agreement with the previously determined K_m of 0.5 mM (75°C) in a P . furiosus extract (16) . The kinetic parameters of the purified FBPase determined at 50°C were as follows: a K_m of 0.31 ± 0.06 mM, a V_max of 0.72 ± 0.04 U/mg, and a catalytic efficiency of 1.12 s^-1 mM^-1 . Thus, the P . furiosus FBPase clearly is a thermoactive enzyme with affinities for fructose-1,6-bisphosphate at 50 and 85°C that are similar .

The specific activities of the P . furiosus FBPase for fructose-1,6-bisphosphate and related substrates were determined at 85°C in the standard assay that measures the release of inorganic phosphate . The 1-ml assay mixture contained 50 mM Tris-HCl buffer (pH 8.0; room temperature), 2.5 mM substrate, 10 mM MgCl₂, and 0.02 mg of purified FBPase . At fructose-1,6-bisphosphate concentrations above 10 mM, the enzyme was subjected to substrate inhibition (data not shown) . The highest activity was obtained with fructose-1,6-bisphosphate (12.2 U/mg) . In addition, myo-inositol-1-phosphate, glucose-1-phosphate, and glycerol-2-phosphate could also be dephosphorylated by the enzyme, although the activities for these substrates were relatively low (1.7 to 7.5%) (Table 1) . The recently described I-1-Pase/FBPase from M . jannaschii (MJ0109) also dephosphorylates these substrates but with a higher relative activity (42 to 61%) (20) (Table 1) . The P . furiosus FBPase appeared to be a rather specific phosphatase, since fructose-1-phosphate, fructose-6-phosphate, glucose-6-phosphate, phosphoenolpyruvate (PEP), 5'-AMP, 5'-ADP, and 5'-ATP could not be used as substrates under the tested conditions .

 
The low I-1-Pase activity of the P . furiosus FBPase might be explained as follows . In thermophilic archaea and bacteria, several intracellular solutes are accumulated in response to osmotic and temperature stresses (15) . One of these compatible solutes is di-myo-inositol phosphate (DIP), a solute that accumulates at supraoptimal growth temperatures in some thermophilic species (15, 17) . In P . furiosus, temperatures above the growth optimum also lead to a significant increase of this compound (11) . Two different routes for DIP synthesis are known: (i) in Methanococcus igneus (closely related to M . jannaschii), I-1-Pase activity is required to form myo-inositol, which acts as a precursor in DIP biosynthesis (4), and (ii) in Pyrococcus woesei, DIP is synthesized in a different way, without the myo-inositol-forming step (17) . This latter pathway includes the coupling of two myo-inositol-1-phosphates without a preceding I-1-Pase-mediated dephosphorylation of one of the myo-inositol-1-phosphate moieties . Since P . furiosus is closely related to P . woesei, it is most likely that in P . furiosus, I-1-Pase activity is not required for DIP synthesis either, which would be in good agreement with the observed low activity of the P . furiosus FBPase on myo-inositol-1-phosphate .

Effectors of FBPase. The effect of inhibitors on the activity of the P . furiosus FBPase was investigated by adding cations and metabolites (0 to 100 mM) to the standard enzyme assay mixture (85°C) (Table 2) . The enzyme has an absolute requirement for Mg²⁺ (data not shown) . The inhibition characteristics of the P . furiosus FBPase clearly differ from those of characterized eukaryal and bacterial FBPases as well as from those of the other presently characterized archaeal I-1-Pase/FBPase homologs . FBPase I from E . coli is very sensitive to AMP and PEP (1) . FBPase II from E . coli is strongly inhibited by ATP and ADP, whereas AMP has no effect on the enzyme activity . Furthermore, FBPase II activity is enhanced in the presence of PEP (6) . PEP also affects FBPase III activity, i.e., inhibition by AMP is reduced when PEP is present (7) . The P . furiosus FBPase was inhibited by ADP and ATP (and to some extent by AMP), but PEP (up to 100 mM) did not influence FBPase activity at all . Therefore, PEP presumably is not an important metabolite in the regulation of FBPase in P . furiosus. In addition, glucose-6-phosphate significantly reduced P . furiosus FBPase activity in vitro (Table 2) .

 
Li⁺ is generally a strong inhibitor of FBPase activity (K_i 0.3 mM) (24) . Under the test conditions we used, Li⁺ significantly reduced the P . furiosus FBPase activity (50% inhibitory concentration [IC₅₀], 1 mM) (Table 2), whereas the addition of Na⁺ and K⁺ produced no effect . Previously, it was shown that I-1-Pases are also strongly inhibited by Li⁺ (IC₅₀ 0.3 mM) (12) . These enzymes have a structural fold similar to that of FBPases (27), and both are members of the sugar phosphatase superfamily (http://scop.mrc-lmb.cam.ac.uk/scop) . Inhibition of mammalian I-1-Pase by Li⁺ is of particular interest, since this enzyme is expressed in brain tissue and forms the main target in medical treatment of manic-depression (14) . The mechanisms of the Li⁺ inhibition of FBPases and IMPases are believed to be essentially the same: Li⁺ binds at one of the metal binding sites, thereby retarding turnover or phosphate release (9, 24) . The residues that constitute this metal binding site are conserved in lithium-sensitive I-1-Pase and in FBPase (Fig . 1) . Remarkably, Li⁺ does not have such a strong effect on the T . maritima (TM1415) and M . jannaschii (MJ0109) enzymes (IC₅₀s, 100 mM for TM1415 and >250 mM for MJ0109), although residues constituting the Li⁺ binding site are conserved (Fig . 1) (9) . Minor variations will probably distinguish the inhibitory effect of Li⁺ on the I-1-Pase from that on FBPase (9) .

Classification of FBPases. Recently, a new classification of bacterial FBPases into three groups (FBPase I, FBPase II, and FBPase III) has been proposed (6) . Eukaryal FBPases are orthologous to the bacterial FBPase I enzymes, since both contain typical FBPase domains (http://www.expasy.ch) and display no I-1-Pase activity (20) . The typical FBPase domain is absent in the bacterial FBPase II and FBPase III enzymes (Table 3), suggesting that they are phylogenetically unrelated to FBPase I enzymes . Remarkably, typical I-1-Pase domains (IMP 1) are also present in the eukaryal FBPase and the bacterial FBPase I enzymes (http://www.expasy.ch) . Bacterial and eukaryal I-1-Pases contain two specific domains (IMP 1 and IMP 2) and, together with the eukaryal FBPase and bacterial FBPase I enzymes, belong to the sugar phosphatase superfamily (http://scop.mrc-lmb.cam.ac.uk/scop) . Comparison of the primary structure of the P . furiosus FBPase with the FBPase and IMP family signatures revealed that this enzyme contains both I-1-Pase domains (IMP 1 and IMP 2) . No obvious FBPase domain could be detected in the P . furiosus sequence (Table 3) (Fig . 1) . The P . furiosus FBPase is homologous to M . jannaschii MJ0109, A . fulgidus AF2372, and T . maritima TM1415, with all three enzymes having IMP 1 and IMP 2 domains present in their primary structures (Fig . 1) and possessing dual activity (i.e., FBPase and I-1-Pase activity) (20) . Since these FBPases display limited sequence identity with both eukaryal and mesophilic bacterial FBPases (12 to 16% identity with FBPase I and 11 to 15% identities with FBPase II and FBPase III) but seem to be significantly related to the I-1-Pases (16 to 35% identity), we propose on the basis of sequence identity and substrate specificity that the P . furiosus FBPase and its homologs constitute a new FBPase family, type IV FBPases (FBPase IV), which is present in euryarchaeal and hyperthermophilic bacterial species and is potentially involved in gluconeogenesis . The presence of conserved domains (IMP 1) in type I and type IV FBPases and I-1-Pases, as well as the similar structural folds of these enzymes (20, 27), suggests that these enzymes share the same phylogenetic origin, as has been suggested previously (20, 27) . It is tempting to speculate that the FBPase IV enzymes originally belonged to the I-1-Pase family and subsequently evolved to efficiently convert fructose-1,6-bisphosphate for function in gluconeogenesis .

 
Nucleotide sequence accession number. The P . furiosus fbpA nucleotide and amino acid sequence data reported in this study have been submitted to GenBank under accession no . AF453319 .

This work was supported by the Earth and Life Sciences foundation (ALW), which is subsidized by The Netherlands Organization for Scientific Research (NWO) .

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