A phosphofructokinase homolog from Pyrobaculum calidifontis displays kinase activity towards pyrimidine nucleosides and ribose 1-phosphate.

The genome of the hyperthermophilic archaeon Pyrobaculum calidifontis contains an open reading frame Pcal_0041 annotated as PfkB family ribokinase consisting of phosphofructokinase and pyrimidine kinase domains. Among biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) from Aeropyrum pernix, which displayed kinase activity towards a broad spectrum of substrates including sugars, sugar phosphates and nucleosides, and 36% identical to a phosphofructokinase from Desulfurococcus amylolyticus In order 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, highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. 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-free 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 P. calidifontis The enzyme is structurally distinct to previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.