Characterization of a bifunctional HPr kinase/phosphorylase from Leuconostoc mesenteroides SY1

Abstract

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 Mg2+ and Mn2+ 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. 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).