Research Article | Spotlight

A Phosphofructokinase Homolog from Pyrobaculum calidifontis Displays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

Iram Aziz, Tahira Bibi, Naeem Rashid, Riku Aono, Haruyuki Atomi, Muhammad Akhtar
William W. Metcalf, Editor
DOI: 10.1128/JB.00284-18

ABSTRACT

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1 · s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts of P. calidifontis indicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) in Thermococcus kodakarensis that functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.

IMPORTANCE The discovery of the pentose bisphosphate pathway in Thermococcus kodakarensis has clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase in Pyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

INTRODUCTION

Sugars are a major carbon source utilized by heterotrophic organisms and their phosphorylation is a key step in cellular metabolism. Sugar phosphorylation is catalyzed by at least three different nonhomologous protein families: the hexokinase family, the galactokinase family, and the ribokinase family (1). Members of the ribokinase family catalyze the phosphorylation of a variety of compounds, including ribose, fructose, nucleosides, and some sugar phosphate molecules such as fructose phosphates (1). In this family, phosphofructokinase 2 (Pfk-2) from Escherichia coli is one of the most studied enzymes and is regarded as a representative member of the PfkB subfamily (2). In addition to the PfkB subfamily, phosphofructokinases also include two other subfamilies, PfkA and PfkC (3, 4).

The glycolytic pathways of some hyperthermophilic archaea display reactions and enzymes that are distinct from those found in the classical pathways in bacteria and eukaryotes (5). One of the features lies in the conversion from glyceraldehyde 3-phosphate (GAP) to 3-phosphoglycerate, where GAP:ferredoxin oxidoreductase and/or nonphosphorylating GAP dehydrogenase catalyzes the conversion in a single step instead of using GAP dehydrogenase and phosphoglycerate kinase in the classical two-step reaction (6–10). Another distinction can be found in the sugar kinases (11). In a number of archaea, the classical ATP-dependent glucokinases and phosphofructokinases (PFKs) are replaced by ADP-dependent glucokinases (12–14) and ADP-dependent PFKs (3, 15–17) or, in the case of Methanocaldococcus jannaschii, a bifunctional ADP-dependent gluco/phosphofructokinase (4). In addition, a pyrophosphate-dependent PFK has been identified in Thermoproteus tenax (18). Owing to the diverse features of archaeal PFKs, structurally related proteins from a number of hyperthermophilic archaea have been examined, including enzymes from Aeropyrum pernix (19, 20), M. jannaschii (21), Desulfurococcus amylolyticus (22), and Thermoplasma acidophilum (23). The proteins displayed distinct substrate specificities, suggesting that these structurally related proteins play different roles in their respective organisms. Although the genus Pyrobaculum is a major genus of the Crenarchaeota and provides a number of fully sequenced genomes, there is no report on PFK-related proteins from this genus.

We have been studying various enzymes from the hyperthermophilic archaeon Pyrobaculum calidifontis (24–29). The genome of P. calidifontis harbors two open reading frames, Pcal_0041 and Pcal_1743, whose products belong to the PfkB family of ribokinases and contain PFK and pyrimidine kinase domains. Here, we report the biochemical characterization of the Pcal_0041 protein, which exhibited a poor PFK activity but relatively high kinase activity toward ribose 1-phosphate (R1P) and pyrimidine nucleosides.

RESULTS

Primary structure of a putative PFK encoded by Pcal_0041.The genome of P. calidifontis contains an open reading frame, Pcal_0041, which is annotated as encoding a PfkB domain-containing protein/possible phosphofructokinase. The open reading frame consisted of 915 nucleotides, encoding a polypeptide of 304 amino acids with a calculated molecular mass of 32,136 Da and a pI of 6.47. The protein was most similar (78% identical) to an uncharacterized ribokinase from Pyrobaculum islandicum (WP_011762863). Among the biochemically characterized enzymes, it was 37% identical to the PfkB from A. pernix (Ape_0012) (19) and 36% identical to a 6-phosphofructokinase from D. amylolyticus (WP_042667458) (22). A sequence alignment among biochemically characterized proteins with PfkB domains revealed the presence of a glycine-glycine dipeptide, 37GG (Fig. 1), which plays a role in the formation of a closed conformation of the enzyme and substrate sequestration in nucleoside kinases (23, 30). An NXXE motif, reported to bind ATP and Mg2+ (31), was found as 185NRAE in Pcal_0041. An anion hole motif, DTXGXGD, which neutralizes the negative charge accumulated during the phosphate transfer (23, 30), was conserved in Pcal_0041 as 242DTTGAGD. Two residues, a lysine and a glutamate (Lys43 and Glu143 in E. coli ribokinase; accession no. P0A9J7), are commonly found in ribokinases and interact with the C-1 hydroxy group of ribose, a group not present in nucleosides due to the formation of a glycosidic bond with the base (32). Although Pcal_0041 was annotated as a PfkB domain-containing ribokinase, the corresponding Lys and Glu residues were replaced by Ser39 and Thr136, respectively (Fig. 1).

FIG 1
FIG 1

Amino acid sequence alignment of previously characterized PfkB family proteins and the Pcal_0041 protein. Sequences were aligned with Multiple Sequence Alignment at Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/). NCBI accession numbers are as follows: P. calidifontis, ABO07481; T. acidophilum, CAC12009; Burkholderia thailandensis, ABC38537; M. jannaschii, AAB98396; A. pernix, BAA78921; D. amylolyticus, WP_042667458; and E. coli, P0A9J6. The NXXE motif involved in metal binding is indicated with the black box. The glycine-glycine dipeptide is indicated with circles, and the residues involved in formation of the oxyanion hole are indicated by the thick line. Residues involved in substrate specificity are indicated with arrowheads, and conserved residues are indicated with asterisks.

Expression of Pcal_0041 and purification of the recombinant protein.To examine whether the Pcal_0041 protein displays PFK activity, we cloned and expressed the gene in E. coli. Heterologous expression of the gene resulted in the production of the recombinant Pcal_0041 protein in a soluble form. The protein was partially purified by heat treatment at 80°C for 20 min, which resulted in the denaturation and precipitation of heat-labile proteins of the host. Pcal_0041 protein, retained in the soluble fraction, was further purified by anion exchange and gel filtration column chromatography to apparent homogeneity, judged by SDS-PAGE (Fig. 2). The protein eluted through a gel filtration column at a retention volume of 17.5 ml, equivalent to 68 kDa. On the basis of the calculated molecular mass of the protein (32,136 Da), recombinant Pcal_0041 exists in a dimeric form.

FIG 2
FIG 2

Coomassie brilliant blue-stained SDS-polyacrylamide gel showing purified recombinant Pcal_0041 protein. Lane M, protein standards; lane 1, purified Pcal_0041 protein (8 μg) after gel filtration column chromatography.

Using fructose 6-phosphate as a substrate, purified Pcal_0041 protein exhibited only low levels of PFK activity (0.77 μmol · min−1 · mg−1). This prompted us to examine the kinase activity against other substrates. As the Ape_0012 protein from A. pernix displayed activity toward nucleosides, we next examined the activity toward adenosine, guanosine, cytidine, and uridine. Pcal_0041 protein exhibited much higher activity when cytidine or uridine was used as the substrate (12 and 10 μmol · min−1 · mg−1, respectively). In contrast, only negligible amounts of activity were detected with guanosine or adenosine. On the basis of these results, cytidine was selected as the substrate to examine the biophysical properties of the enzyme.

Effect of pH and temperature.The effect of pH was examined by measuring the enzyme activity at 85°C in different buffer solutions of various pHs ranging from 5.5 to 11.0. Recombinant Pcal_0041 protein maintained its activity over a relatively wide pH range (Fig. 3A), and the highest activity was found at pH 8.0 in HEPES-NaOH buffer. The effect of temperature on the enzyme activity was examined at various temperatures between 50 and 90°C. Enzyme activity increased with increases in temperature (Fig. 3B). An Arrhenius plot was made from 50 to 90°C, and the activation energy of the reaction was calculated as 67.2 kJ/mol. The thermostability of Pcal_0041 protein was examined at 100°C. The protein was highly stable with a half-life of 90 min at this temperature (Fig. 3C). The structural stability of recombinant Pcal_0041 was also analyzed by circular dichroism (CD) spectroscopy at different temperatures (30 to 90°C). The CD spectra showed that there was no significant change in the spectrum up to 90°C (Fig. 3D), indicating that the enzyme maintains its secondary structure at least up to 90°C, which is in good agreement with the effects of temperature on enzyme activity (Fig. 3B).

FIG 3
FIG 3

Effects of pH and temperature on the kinase activity of Pcal_0041. (A) Effect of pH. Kinase activity of Pcal_0041 was examined at 85°C in various buffers: ■, MES-NaOH (pH 5.5 to 7.0); □, HEPES-NaOH (pH 7.0 to 8.0); ●, Tricine-NaOH (pH 8.0 to 9.0); ○, CHES-NaOH (9.0 to 10); ▲, CAPS-NaOH (10.0 to 11.0). (B) Effect of temperature. Enzyme activity was examined in HEPES-NaOH buffer (pH 8.0) at various temperatures from 50 to 90°C. (C) Thermostability of Pcal_0041 at 100°C. Pcal_0041 was heated at 100°C for various intervals of time and the residual activity was examined at 85°C and pH 8.0. All buffers were prepared at the temperatures of use. Each point is the average from at least three measurements. SD values are indicated with bars. (D) Circular dichroism studies on recombinant Pcal_0041 protein. CD spectra from 190 to 280 nm were measured at various temperatures: ◆, 30°C; ■, 50°C; ▲, 70°C; ✖, 80°C; ○, 90°C.

Kinase activity toward various substrates.The ATP-dependent kinase activity of Pcal_0041 protein was examined toward various sugars, sugar phosphates, nucleosides, and deoxynucleosides. Pcal_0041 protein utilized a variety of compounds as the substrates (Fig. 4). We included R1P, as an ADP-dependent R1P kinase, a component of the pentose bisphosphate pathway, was recently discovered in Thermococcus kodakarensis (33). The highest activity (15 μmol · min−1 · mg−1) was found when R1P was used as the substrate, followed by cytidine (12 μmol · min−1 · mg−1) and uridine (10 μmol · min−1 · mg−1). All other substrates resulted in specific activities lower than 3 μmol · min−1 · mg−1.

FIG 4
FIG 4

Kinase activity of Pcal_0041 protein towards various substrates. Kinase activity was examined at 85°C and pH 8.0 using various substrates at a concentration of 5 mM. Values are the averages from three measurements, and bars indicate the SD values.

When various phosphoryl donors were examined with cytidine as the cosubstrate, we found that the enzyme displayed a marked preference for ATP (100%), followed by GTP (76%). Negligible amount of activity (3%) was detected with CTP as the phosphate donor (Fig. 5). No activity could be detected with ADP and pyrophosphate. The results suggest that the Pcal_0041 protein is a nucleoside triphosphate-dependent enzyme, distinct from the ADP-dependent R1P kinase of T. kodakarensis.

FIG 5
FIG 5

Comparison of kinase activity in the presence of various phosphoryl donors. Activity was examined using cytidine as the substrate at 85°C and pH 8.0. The concentration of each phosphate donor was 5 mM along with 5 mM MgCl2. Values are the averages from three measurements, and bars indicate the SD values.

Kinetic parameters.The kinetic parameters toward R1P, cytidine, and uridine were measured by varying the concentration of each substrate in the presence of 2 mM ATP. The parameters for ATP were determined by measuring the activity at various concentrations in the presence of 2 mM cytidine. The enzyme followed Michaelis-Menten kinetics, with Km values of 0.71 ± 0.18 mM toward R1P, 1.1 ± 0.2 mM toward uridine, and 2.1 ± 0.36 mM toward cytidine. Vmax values of 15.0 ± 0.7, 12.0 ± 0.6, and 10.5 ± 0.4 μmol · min−1 · mg−1 were calculated when R1P, cytidine, and uridine were used as the substrates, respectively. From the Vmax value toward R1P and molecular mass of the Pcal_0041 protein (32,136 Da), a kcat value of 8.2 s−1 was calculated. The catalytic efficiency (kcat/Km) of the Pcal_0041 protein toward R1P was 11.5 mM−1 · s−1, while values toward cytidine and uridine were 3.1 mM−1 · s−1 and 5.1 mM−1 · s−1, respectively. The relatively high catalytic efficiency toward R1P raises the possibility that Pcal_0041 encodes an R1P kinase.

Production of ribose 1,5-bisphosphate in P. calidifontis cell extracts.As the Pcal_0041 protein exhibited R1P kinase activity, we examined whether R1P was actually converted to R15P in P. calidifontis cell extracts (CFE). R1P was added to CFE along with ATP. R15P formation was confirmed with a coupling assay using ribose-1,5-bisphosphate isomerase (R15P isomerase) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) from T. kodakarensis (33). R15P isomerase catalyzes an isomerization reaction converting R15P to ribulose 1,5-bisphosphate, the substrate of RubisCO. RubisCO converts ribulose 1,5-bisphosphate, CO2, and H2O to two molecules of 3-phosphoglycerate. R1P kinase activity was clearly detected in the extracts (0.17 μmol · min−1 · mg−1). We next examined the presence of nucleoside phosphorylase activity in the cell extracts. A nucleoside phosphorylase activity would provide R1P from nucleosides and phosphate. When a mixture of nucleosides (10 mM each) was added to CFE along with 20 mM phosphate, we observed the formation of R1P. The activity level corresponded to 0.16 μmol · min−1 · mg−1. The results indicate the presence of a pathway in P. calidifontis that converts the ribose moieties of nucleosides to R15P. We also examined the presence of R15P isomerase activity in the cell extracts. As described above, R1P was added to cell extracts along with ATP. In this case however, R15P isomerase was excluded from the coupling assay so that ribulose 1,5-bisphosphate would have to be generated from R15P by an endogenous R15P isomerase in the cell extract. In this case however, we could not detect the generation of ribulose 1,5-bisphosphate in P. calidifontis extracts. AMP phosphorylase activity, which would generate R15P and adenine from AMP and phosphate, was also not detected in the CFE.

DISCUSSION

This study revealed that Pcal_0041 from P. calidifontis encodes a protein with pyrimidine nucleoside kinase activity and R1P kinase activity. As for the phosphate donor, the highest activity was found in the presence of ATP followed by GTP. Among archaea, a number of members of the ribokinase family have recently been characterized and have been shown to display diverse substrate specificities. In euryarchaeota, a ribokinase from M. jannaschii (MJ0406) is an ATP-dependent nucleoside kinase that prefers guanosine, cytidine, and inosine (21). In T. acidophilum, the Ta0880 protein is also a nucleoside kinase with broad substrate specificities, with the highest activity with guanosine (23). In T. kodakarensis, the TK2285 protein is a myo-inositol kinase (34), the TK2029 protein is an ADP-dependent R1P kinase, and the protein encoded by TK1843 is an ATP-dependent cytidine kinase (33). ADP-dependent glucokinases which belong to the ribokinase superfamily have been identified in organisms, including Pyrococcus furiosus (12), Thermococcus litoralis (EHR77687) (35), and Archaeoglobus fulgidus (14). The ADP-dependent glucokinase in T. kodakarensis also acts as an ADP-dependent glucosamine kinase in chitin degradation (36). As described above, among crenarchaeotes, ribokinases from A. pernix (Ape_0012) and D. amylolyticus (WP_042667458) have been characterized (19, 22). The Ape_2091 protein, another ribokinase from A. pernix, is an ATP-dependent glucokinase, but it can also phosphorylate glucosamine, fructose, mannose, and 2-deoxyglucose (37). AJ510140 from T. tenax encodes an ATP-dependent hexokinase (38).

The substrate specificity of Pcal_0041 is quite intriguing, showing the highest catalytic activity with R1P. However, with the exception of the TK2029 protein from T. kodakarensis, which is an ADP-dependent R1P kinase, none of the ribokinases from the archaea have actually been examined with R1P as a potential substrate. The TK2029 protein from T. kodakarensis is 34% identical to Pcal_0041. To examine whether substrate specificity can be distinguished by primary structure, we constructed a phylogenetic tree with ribokinase proteins from the Archaea (Fig. 6). The tree includes biochemically characterized ribokinase proteins from archaea, along with all of their homologs with at least 30% identity to the Pcal_0041 protein. To reduce the number of total proteins in the tree, homologs were limited to those from the following species: A. fulgidus DSM 4304, A. pernix K1, D. amylolyticus DSM 16532, D. amylolyticus Z-533, M. jannaschii, Picrophilus torridus, Pyrobaculum aerophilum, P. calidifontis VA1, P. furiosus DSM 3638, Pyrococcus horikoshii OT3, Sulfolobus solfataricus, Sulfolobus acidocaldarius DSM 639, T. kodakarensis KOD1, Thermococcus onnurineus NA1, T. acidophilum, T. litoralis, Thermococcus zilligii, and T. tenax. Although some clades may be further divided as our knowledge increases, we can observe 10 clades or groups (groups 1 to 10) in the tree shown in Fig. 6. Groups 1 through 6 have at least two members that have been experimentally examined and suggest that the members of each group display the same activity. Groups 3 and 6 represent the ADP- and ATP-dependent gluco/hexokinases, respectively, while groups 2 and 4 consist of the ADP- and ATP-dependent phosphofructokinases, respectively. We cannot predict the functions of Tli_OCC03492 and Pto_PTO1216 with confidence, as Tli_OCC03492 is a lone member in group 1 from euryarchaeota that otherwise consists only of proteins from Crenarchaeota. Pto_PTO1216 represents a deeply diverging branch from group 2. On the other hand, the function of Sai_Saci0272 from S. acidocaldarius will also need further investigation, as other members of this group derive from Euryarchaeota. Groups 7 through 10 have only a single member that has been biochemically characterized, and further verification will be needed to support the functions of these groups. However, the tree clearly indicates that the Pcal_0041 protein represents a previously uncharacterized group of archaeal ribokinase family proteins, which may function as ATP-dependent R1P kinases. Although the catalytic efficiencies support a role as an R1P kinase, at present we cannot rule out the possibility that the protein functions as a nucleoside kinase. The characterization of other members of group 10 or the development of a genetic system in P. calidifontis and subsequent genetic analyses will be necessary to clarify the metabolic role of the protein.

FIG 6
FIG 6

A phylogenetic tree of ribokinase family proteins from the Archaea. Ribokinase family proteins that have been experimentally characterized were used to retrieve homologs from 18 selected archaeal species. Proteins that displayed at least 30% identity are included in the analysis. Proteins from Crenarchaeota are indicated in red, and those from Euryarchaeota are indicated in black. The organisms are abbreviated as follows: Afu, A. fulgidus DSM 4304; Ape, A. pernix K1; DamA, D. amylolyticus DSM 16532; DamB, D. amylolyticus Z-533; Mja, M. jannaschii DSM 2661; Pto, Picrophilus torridus DSM 9790; Pae, Pyrobaculum aerophilum IM2; Pca, P. calidifontis VA1; Pfu, P. furiosus DSM 3638; Pho, Pyrococcus horikoshii OT3; Sso Sulfolobus solfataricus P1; Sai, Sulfolobus acidocaldarius DSM 639; Tko, T. kodakarensis KOD1; Ton, Thermococcus onnurineus NA1; Tac, T. acidophilum DSM 1728; Tli, T. litoralis DSM 5473; Tzi, Thermococcus zilligii AN1; Tte, and T. tenax kra1. The tree was constructed with Phyml at Phylogeny.fr after aligning the sequences with MUSCLE.

A wider search indicated that there are homologs in bacteria that also display over 30% identity with the archaeal Pcal_0041 protein. None of the proteins have been experimentally evaluated. Adding these to the phylogenetic tree indicated that they fall into three groups (see Fig. S1 in the supplemental material). The addition of the bacterial sequences results in a division of the archaeal groups 5 and 6. Among the bacterial sequence groups, one is related to Sai_Saci0272 and the other two are distantly related with the ATP-dependent hexokinases. Members of group 10, which includes the Pcal_0041 protein, still cluster in the expanded tree.

Nucleoside degradation has been shown to be carried out by the following pathway(s), depending on the organism. Nucleoside phosphorylase catalyzes the phosphorolysis reaction of nucleosides, releasing the base and generating R1P. Phosphoribomutase, or phosphopentomutase, converts R1P to ribose 5-phosphate (R5P). R5P can enter the pentose phosphate pathway and is directed to central carbon metabolism. R5P can also be converted to phosphoribosyl pyrophosphate (PRPP) and be used as starting material for the biosynthesis of a wide range of molecules such as nucleotides and amino acids. These routes are widely conserved in bacteria and eukaryotes. In the archaea, genes for nucleoside phosphorylase homologs are present in their genomes, suggesting that R1P is generated in archaea. This has been experimentally demonstrated in T. kodakarensis and in this study on P. calidifontis. In many archaea however, clear homologs are not present for phosphoribomutase and enzymes of the pentose phosphate pathway. On the other hand, an ADP-dependent R1P kinase, which converts R1P to R15P, is present in members of the Thermococcales. These organisms also harbor an R15P isomerase, generating ribulose 1,5-bisphosphate (RuBP), and RubisCO, which converts RuBP, carbon dioxide, and water to 3-phosphoglycerate. The genome of P. calidifontis contains three open reading frames, namely, Pcal_0435, Pcal_0814, and Pcal_1837, which are annotated as encoding nucleoside phosphorylases, and they may be responsible for the nucleoside phosphorylase activity in P. calidifontis detected in this study. There is no clear homolog of phosphoribomutase, as in the case in T. kodakarensis. The activity of the Pcal_0041 protein would be able to convert R1P to R15P. Intriguingly, in P. calidifontis, an R15P isomerase homolog (as well as a RubisCO homolog) could not be found. Furthermore, in our initial activity tests, we could not detect R15P isomerase activity in the cell extracts of P. calidifontis. R15P has been reported to be an activator of phosphofructokinase and inhibitor of fructose-1,6-bisphosphatase in mammalian cells (39). Although we cannot rule out the possibility that R15P acts as a regulator of glycolysis/gluconeogenesis in P. calidifontis, this is unlikely, as the structures and reaction mechanisms of archaeal fructose-1,6-bisphosphatases differ completely from those of the mammalian enzymes (40–44). Furthermore, a regulatory role of R15P does not explain how nucleosides are degraded in this archaeon. Adenosine is a product of the ubiquitous S-adenosylmethionine methylation mechanism, and a pathway for its degradation and/or use should be present in all organisms. There is a possibility that the Pcal_0041 protein functions as a nucleoside kinase in P. calidifontis, as significant levels of activity were observed with cytidine and uridine. However, we feel that this is unlikely, as we clearly observe the generation of R1P in P. calidifontis with the addition of nucleosides and phosphate. There are a few possibilities that should be explored in future studies. One is that although we did not observe R15P isomerase activity in P. calidifontis cell extracts, this may be due to the absence of an activator other than AMP or dAMP, which activates the R15P isomerase from T. kodakarensis (45). Another possibility is the presence of R15P kinase activity, which would convert R15P to phosphoribosylpyrophosphate (PRPP). Although AMP phosphorylase activity was not observed in the cell extracts, a third possibility would be an NMP phosphorylase activity. This would directly convert R15P and a base to a nucleoside monophosphate and phosphate. This activity is present in T. kodakarensis for the bases adenine, cytosine, and uracil (45). Although further investigations are needed to establish the physiological role of Pcal_0041 and determine the fate of R15P, our results suggest that the ribose moieties of nucleosides can be converted to R15P in P. calidifontis via nucleoside phosphorylase(s) and an ATP-dependent R1P kinase encoded by Pcal_0041.

MATERIALS AND METHODS

Strains, media, and culture conditions.P. calidifontis is a facultatively aerobic, heterotrophic, hyperthermophilic archaeon isolated from a terrestrial hot spring in the Philippines (25). Its complete genome has been sequenced and annotated (http://www.ncbi.nlm.nih.gov/nuccore/CP000561.1). P. calidifontis was grown aerobically in TY medium consisting of 10 g · liter−1 tryptone, 1 g · liter−1 yeast extract, and 3 g · liter−1 sodium thiosulfate. E. coli strains DH5α and BL21-CodonPlus(DE3)-RIL were used for plasmid construction and gene expression, respectively, and were cultivated at 37°C in lysogeny broth (LB) containing ampicillin (100 mg · liter−1). Unless mentioned otherwise, all chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan).

Chemicals and enzymes.Adenosine, guanosine, cytidine, uridine, glucose 1-phosphate, glucose 6-phosphate, fructose, fructose 6-phosphate, fructose 1-phosphate, ribose, ribose 5-phosphate, lactose, mannose, xylose, xylulose, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, deoxyribose 1-phosphate, inosine, xanthosine, sedoheptulose 7-phosphate, and N-acetylglucosamine were purchased from Sigma-Aldrich (St. Louis, MO). R1P was purchased from Toronto Research Chemicals (Toronto, Canada). Phosphoenolpyruvate, galactose, and glucose were purchased from Wako Pure Chemicals Limited (Osaka, Japan). ATP and NADH were purchased from Oriental Yeast (Tokyo, Japan). Tris, N,N-bis(2-hydroxyethyl)glycine (Bicine), 2-(N-morpholino)ethanesulfonic acid (MES), 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine), 2-(cyclohexylamino)ethanesulfonic acid (CHES), lactose, 2-deoxyribose, myo-inositol, AMP, and dAMP were purchased from Nacalai Tesque. Pyruvate kinase/lactate dehydrogenase (PK/LDH) from rabbit muscle, 3-phosphoglyceric phosphokinase from baker's yeast, glyceraldehyde 3-phosphate dehydrogenase from rabbit muscle, triosephosphate isomerase from baker's yeast, and α-glycerophosphate dehydrogenase from rabbit muscle were purchased from Sigma-Aldrich.

Cloning of the Pcal_0041 gene.To isolate genomic DNA from P. calidifontis, cells grown in TY medium (90°C for 48 h at 150 rpm shaking) were collected by centrifugation (10,000 × g at 4°C for 15 min) and resuspended in 20 ml of TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 10 mM NaCl [pH 8.0]). After a second centrifugation, the pellet was washed with 10 ml of SET buffer (20% sucrose, 50 mM Tris-HCl, 50 mM EDTA [pH 8.0]). Cells were lysed by adding 1 ml of lysozyme solution (5 mg · ml−1 lysozyme in TEN buffer) and incubating for 40 min at 37°C. Following this, 10 ml of TEN buffer, 0.5 ml of SDS solution (250 mg · ml−1), and 2.5 μl proteinase K solution (0.2 mg · ml−1) were added, and the mixture was incubated at 60°C for 15 min. After cooling, an equal volume of buffered phenol (0.1 M Tris-HCl, pH 7.6) and chloroform (1:1 ratio) was added. The solution was mixed gently and centrifuged. The aqueous layer was subjected to phenol-chloroform treatment again, and the DNA in the aqueous layer was precipitated by adding a 2-fold volume of chilled absolute ethanol and cooling for 1 h at −20°C. The precipitated genomic DNA was retrieved with a sterile glass dropper and washed with ice-cold 70% (vol/vol) ethanol. After centrifugation, the DNA pellet was dried at room temperature and dissolved in 500 μl of deionized water. The solution was treated with RNase (50 μg · ml−1) for 30 min at 37°C. The DNA was analyzed by 1% agarose gel electrophoresis and the concentration was calculated by measuring the absorbance at 260 nm.

The open reading frame of Pcal_0041 was amplified by PCR using the primers Pcal_0041F (5′-CATATGTTAGTGGCCTCTGTCGGCAACC-3′, NdeI site is underlined) and Pcal_0041R (5′-CCTCTGCACTGTTTAAACTACTC-3′) and genomic DNA of P. calidifontis. The amplified DNA fragment was cloned in the pTZ57R/T cloning vector using T4 DNA ligase, resulting in plasmid pTZ-0041. To insert the gene into the expression vector, pET-21a, the gene was digested from pTZ-0041 using NdeI (introduced in the forward primer) and HindIII (present in the multiple cloning site of pTZ57R) and inserted into pET-21a expression vector digested with the same enzymes. The resulting plasmid was named pET-0041.

Production of recombinant Pcal_0041 protein.For the production of recombinant Pcal_0041 protein, E. coli BL21-CodonPlus(DE3)-RIL cells were transformed with pET-0041. The transformed cells were cultivated in LB to an optical density of 0.4 to 0.5 at 660 nm. Gene expression was then induced by the addition of isopropyl-β-d-1-thiogalactopyranoside at a final concentration of 0.2 mM, and the cells were further incubated for 4 h at 37°C. The cells were harvested by centrifugation (1 g wet weight from a 500-ml culture), resuspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM β-mercaptoethanol, and disrupted by sonication. Soluble and insoluble fractions were separated, and the supernatant containing recombinant Pcal_0041 protein was heated at 80°C for 30 min to denature heat-labile proteins from E. coli. The denatured proteins were removed by centrifugation at 20,000 × g for 20 min. The supernatant containing Pcal_0041 protein was loaded onto a Resource Q (GE Healthcare, Buckinghamshire, UK) anion exchange column, and proteins were eluted with a 0 to 1 M NaCl gradient in 50 mM Tris-HCl (pH 7.5). The fractions containing Pcal_0041 protein were pooled and applied to a Superdex 200 10/300 GL gel filtration column (GE Healthcare) equilibrated with 150 mM NaCl in 50 mM Tris-HCl (pH 7.5).

The molecular mass of recombinant Pcal_0041 protein was determined by SDS-PAGE analysis as well by gel filtration chromatography. The standard curve was obtained with ferritin (440 kDa), catalase (240 kDa), lactate dehydrogenase (140 kDa), bovine serum albumin ([BSA] 64.5 kDa), and proteinase K (28.9 kDa). Solutions of the standard and sample proteins were prepared in 20 mM Tris-HCl (pH 8.0) containing 150 mM NaCl. Protein concentration was determined spectrophotometrically with Bradford reagent using BSA as a standard.

Kinase activity measurements on the recombinant Pcal_0041 protein.Enzyme assays for kinase activity were carried out by coupling the reaction with pyruvate kinase/lactate dehydrogenase (PK/LDH), and the amount of ADP formed was measured by monitoring the oxidation of NADH. Discontinuous enzyme assays were used for all reactions (33). The first reaction was carried out in a 100-μl reaction mixture containing 5 mM substrate, 5 mM ATP, 5 mM MgCl2, and 10 mM KCl in 50 mM HEPES buffer (pH 8.0) and purified Pcal_0041 protein. Routine assays were performed at 85°C with a 2-min incubation time and immediately terminated by quenching on ice. Enzyme from the first reaction mixture was removed by Amicon Ultra centrifugal filter unit (molecular weight cutoff [MWCO], 10,000). The supernatant obtained from filtration was used for the second reaction in which the generation of ADP from ATP in the first reaction was quantified with the PK/LDH reaction. The PK/LDH reaction mixture consisted of 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.2 mM NADH, 5 mM phosphoenolpyruvate, 50 U/ml pyruvate kinase, and 70 U/ml of lactate dehydrogenase. The decrease in absorbance at 340 nm was measured. The results of control experiments without phosphate acceptor were subtracted. This method was also applied for examining the nucleotide specificity of Pcal_0041 protein. ATP, UTP, GTP, and CTP were used as the phosphate donors and cytidine as the phosphate acceptor.

Reaction products after the first reaction were also analyzed by high-pressure liquid chromatography (HPLC) using a COSMOSIL C18-PAQ column (Nacalai Tesque) at 40°C with 50 mM NaH2PO4 (pH 4.3) as a mobile phase. The absorption was measured at 254 nm by using a UV detector. Standards, AMP, ADP, ATP, cytidine, and CMP, were applied at a final concentration of 5 mM.

Effects of pH and temperature.The effect of pH on the enzyme activity of Pcal_0041 protein was measured at 85°C using the following buffers: MES-NaOH buffer (pH 5.5 to 7.0), HEPES-NaOH buffer (pH 7.0 to 8.0), Tricine-NaOH buffer (pH 8.0 to 9.0), CHES-NaOH buffer (pH 9.0 to 10.0), and CAPS-NaOH buffer (pH 10.0 to 11.0). All buffers were prepared at 85°C. The effect of temperature was measured by varying the temperature from 50 to 90°C at pH 8.0 in HEPES buffer. All buffers were prepared at the respective temperatures. Thermostability was examined by incubating the recombinant protein at 100°C in 50 mM HEPES buffer (pH 8.0) for various intervals of time and measuring the residual activity at 85°C. All measurements were carried out in triplicates.

Determination of kinetic parameters.Kinetic analyses were carried out towards cytidine, uridine, and R1P. All reactions were carried out at 85°C with a 2-min incubation time. Substrate concentrations were varied from 0 to 15 mM.

Analysis of enzyme activities in P. calidifontis cell extracts.Cell extracts (CFE) of P. calidifontis were prepared as follows. Cells cultivated in TY medium (90°C for 48 h at 150 rpm shaking) were harvested by centrifugation (10,000 × g for 15 min at 4°C), suspended in 50 mM Tris-HCl (pH 7.5), and disrupted by sonication. The supernatant after centrifugation (20,000 × g for 30 min at 4°C) was used as the CFE.

R1P kinase activity.The formation of R15P was examined by coupling assays with R15P isomerase and RubisCO from T. kodakarensis (33). The composition of the kinase reaction was as described above, using CFE instead of purified recombinant protein. After removing the proteins with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), the second reaction was carried out in a 100-μl reaction mixture containing 100 mM NaHCO3, 5 μg RubisCO, 5 μg R15P isomerase, 5 mM AMP, 5 mM MgCl2, and an aliquot of the kinase reaction mixture. The incubation was carried out at 85°C for 10 min and then immediately quenched on ice. Enzymes were removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), and the filtrate was used to measure 3-phosphoglyceric acid (3-PGA) formation. 3-PGA was quantified in a reaction mixture containing 5 mM ATP, 0.2 mM NADH, 160 mM Bicine-NaOH buffer containing 16 mM MgCl2, phosphoglycerate phosphokinase (563 U/ml), glyceraldehyde 3-phosphate dehydrogenase (125 U/ml), triose phosphate isomerase (260 U/ml), glycerophosphate dehydrogenase (22.5 U/ml), and 5 mM reduced glutathione. The reaction mixture was preincubated at 25°C for 3 min. The decrease in absorbance at 340 nm was measured. For each measurement, an assay mixture without R1P in the first kinase reaction was used as a control and its absorbance was subtracted (33).

AMP phosphorylase activity.The formation of R15P from AMP and phosphate was examined with the same coupling assays used to detect R1P kinase activity. In the first reaction (conversion of AMP to R15P), 30 mM sodium phosphate (pH 7.5) and 100 μg CFE in 100 mM Tris-HCl (pH 7.5) were preincubated for 5 min at 85°C, followed by the addition of 30 mM AMP. After 10 min of incubation at 85°C, the mixture was quenched on ice and filtered using an Amicon Ultra centrifugal filter unit (MWCO, 10,000). For each measurement, an assay mixture without CFE was used as a control and its absorbance was subtracted.

Nucleoside phosphorylase activity.The first reaction mixture (conversion of nucleosides to R1P) contained 10 mM (each) cytidine, uridine, adenosine, and guanosine, 100 μg CFE, and 20 mM sodium phosphate. The reaction mixture was incubated for 5 min at 85°C and quenched on ice. Proteins were removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), and the filtrate was used for the second reaction. The second reaction (conversion of R1P to R15P) contained 5 mM ATP, 5 mM MgCl2, 10 mM KCl, and 5 μg Pcal_0041 protein in 50 mM HEPES-NaOH buffer (pH 8.0). The reaction mixture was preincubated at 85°C for 5 min, and the reaction was started by adding an aliquot (30 μl) from the first reaction. After 2 min of incubation at 85°C, the assay mixture was quenched on ice. Protein was removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), and the formation of R15P was quantified with the methods described above. For each measurement, an assay mixture without CFE was used as a control and its absorbance was subtracted.

R15P isomerase activity.The first reaction was carried out in a 100-μl reaction mixture containing 5 mM ATP, 5 mM MgCl2, 5 mM R1P, 10 mM KCl, 100 μg CFE, 100 mM NaHCO3, 5 μg RubisCO, and 5 mM AMP in 50 mM HEPES buffer (pH 8.0). A mixture without R1P was incubated at 85°C for 5 min, and the reaction was started by the addition of 5 mM R1P. After a 2-min incubation, the reaction mixture was quenched on ice and the proteins were removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000). 3-PGA in the filtrate was quantified as described above. For each measurement, an assay mixture without R1P was used as a control and its absorbance was subtracted.

The presence of R15P isomerase activity was also examined using nucleosides as the substrates. The first reaction mixture (conversion of nucleosides to R1P) contained 10 mM nucleosides (cytidine, uridine, adenosine, and guanosine), 100 μg CFE, and 20 mM sodium phosphate. The reaction mixture was incubated for 5 min at 85°C and quenched on ice. Proteins were removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), and the filtrate was used for the next reaction. The second reaction (conversion of R1P to 3-PGA via R15P and RuBP) contained 5 mM ATP, 5 mM MgCl2, 10 mM KCl, 100 mM NaHCO3, 5 mM dAMP, 5 μg Tk-RubisCO, and 5 μg Pcal_0041 protein in 50 mM HEPES-NaOH buffer (pH 8.0). The reaction mixture was preincubated at 85°C for 5 min, and the reaction was started by adding an aliquot (30 μl) of the first reaction. After 2 min of incubation at 85°C, the assay mixture was quenched on ice. The enzymes from the assay mixture were removed with an Amicon Ultra centrifugal filter unit (MWCO, 10,000), and the filtrate was used for the third reaction. 3-PGA formation was measured as described above for R15P formation. For each measurement, an assay mixture without nucleosides was used as a control.

Circular dichroism analysis.The structural stability of Pcal_0041 protein was analyzed by circular dichroism (CD) spectroscopy using a Chirascan-plus CD spectrometer (Applied Photophysics, UK). The protein samples were incubated at different temperatures ranging from 50 to 90°C. The CD spectra of the protein solutions were recorded in 20 mM Tris-HCl (pH 8.0) in the far UV range of 200 to 260 nm. Solvent spectra were subtracted from those of the protein solution.

ACKNOWLEDGMENTS

This study was partially funded by the Core Research for Evolutional Science and Technology program of the Japan Science and Technology Agency to H.A. within the research area “Creation of Basic Technology for Improved Bioenergy Production through Functional Analysis and Regulation of Algae and Other Aquatic Microorganisms.” This study was also partially supported by grant no. 20-2024 to N.R. by the Higher Education Commission of Pakistan.

FOOTNOTES

    • Received 6 May 2018.
    • Accepted 17 May 2018.
    • Accepted manuscript posted online 4 June 2018.
  • Address correspondence to Naeem Rashid, naeem.ff.sbs{at}pu.edu.pk, or Haruyuki Atomi, atomi{at}sbchem.kyoto-u.ac.jp.

  • Citation Aziz I, Bibi T, Rashid N, Aono R, Atomi H, Akhtar M. 2018. A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate. J Bacteriol 200:e00284-18. https://doi.org/10.1128/JB.00284-18.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00284-18.

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KEYWORDS

Pyrobaculum calidifontis
ribokinase
nucleoside kinase
phosphofructokinase
ribose 1-phosphate kinase
Archaea
Pyrobaculum
hyperthermophiles
metabolism
nucleoside
pentose
pentose bisphosphate pathway

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