The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK0376, annotated as ADP-dependent phosphofructokinase belonging to pfkC family. The encoding gene was expressed in Escherichia coli and the gene product was characterized. The recombinant protein was produced in soluble and active form. Phosphofructokinase activity of TK0376 was metal-ion dependent and the highest activity (5090 mu mol min(-1) mg(-1)) was found in the presence of Co2+ followed by Mg2+ (3280 mu mol min(-1) mg(-1)) at 90 degrees C and pH 7.5. TK0376 preferred ADP as phosphoryl donor, however, it could be replaced by ATP but with a 5-fold lower activity. It catalyzed the phosphorylation of fructose 6-phosphate and dephosphorylation of fructose 1,6-bisphosphate. In addition, it was able to phosphorylate glucose and nucleosides but with a much lower rate compared to that of fructose 6-phosphate. The apparent k(cat) and K-m. values against fructose 6-phosphate were 4238 s(-1) and 0.74 mM, respectively. The rate of dephosphorylation of fructose 1,6-bisphosphate was 3-times lower at 50 degrees C than the phosphorylation of fructose 6-phosphate. Similarly, the rate of phosphorylation of glucose was 450-fold lower than that of fructose 6-phosphate. Phosphofructokinase activity was not allosterically regulated, but it was slightly enhanced by phosphoenol pyruvate, and inhibited by ATP and AMP in a competitive manner. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.