Characterization of a Bifunctional HPr Kinase/Phosphorylase from Leuconostoc mesenteroides SY1

The hprK gene encoding bifunctional HPrK/P (kinase/phosphorylase) was cloned from L. mesenteroides SY1, a strain isolated from kimchi. hprK was transcribed as a monocistronic gene. His-tagged HPrH16A and HPrK/P were produced in E. coli BL21 (DE3) using pET26b(+) and purified. HPrK/P phosphorylation assay with purified proteins showed that the kinase activity of HPrK/P increased at slightly acidic pHs. Divalent cations such as $Mg^{2+}$ and $Mn^{2+}$ and glycolytic intermediates such as fructose-1, 6-bisphosphate (FBP) and phosphoenolpyruvate (PEP) increased the kinase activity of HPrK/P, but inorganic phosphate strongly inhibited it. Kinetic studies for the kinase activity of HPrK/P showed that the apparent $K_m$ values were 0.18 and $14.57{\mu}M$ for ATP and HPr, respectively. The $K_m$ value for the phosphorylase activity of HPrK/P was $14.16{\mu}M$ for P-Ser-HPr (HPr phosphorylated at the serine residue).