Structural and functional analyses of Pcal_1233, a highly thermoactive glycerate 2-kinase from Pyrobaculum...

P. calidifontis genome sequence harbours an open reading frame, Pcal_1233, which is annotated as glycerate kinase type-2 family protein. Here, we have verified the kinase activity, substrate and product specificity, and assessed the kinetic parameters of Pcal_1233. Recombinant Pcal_1233 was highly thermoactive displaying an increase in activity with the increase in temperature till 100 °C. Half-life of the enzyme was ~90 min at 100 °C. There were no significant structural changes between 60 and 100 °C as predicted by CD spectroscopy. In addition to temperature, Pcal_1233 was very stable against denaturants. There was no notable loss in kinase activity even after an incubation of 96 h in 8 M urea. Presence of EDTA in the assay mixture abolished the enzymatic activity, indicating that Pcal_1233 is a metal ions dependent enzyme. Among phosphoryl donors, Pcal_1233 was specific towards nucleoside triphosphates, with highest activity against ATP, exhibiting apparent K_m and V_max values of 0.093 ± 0.05 mM and 745 ± 125 μmol min^-1 mg^-1, respectively. Among phosphoryl accepters, it was specific towards glycerate with apparent K_m and V_max values of 0.15 ± 0.06 mM and 685 ± 75 μmol min^-1 mg^-1, respectively. It is most likely that Pcal_1233 selectively produce 2-phosphoglycerate, similar to other Class II glycerate kinases. High enzyme activity and product specificity make Pcal_1233 a potential candidate for enzymatic synthesis of 2-phosphoglycerate.