ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent K values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (k/K) values against these two substrates were 6.2 × 10 ± 0.25 and 5.8 × 10 ± 0.75 M s. The apparent K value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (k/K) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.