Biochemical characterization of a highly active ADP-dependent phosphofructokinase from Thermococcus kodakarensis

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 μmol min−1 mg−1) was found in the presence of Co2+ followed by Mg2+ (3280 μmol min−1 mg−1) at 90°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 kcat and Km 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°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. •TK0376 was produced in soluble and highly active form in E. coli.•TK0376 protein exhibited highest activity in the presence of Co2+.•TK0376 catalyzed the phosphorylation of fructose 6-phosphate as well as dephosphorylation of fructose 1,6-bisphosphate.•TK0376 exhibited highest phosphofructokinase activity among all the characterized counterparts.