The hprK gene of Enterococcus faecalis encodes a novel bifunctional enzyme: the HPr kinase/phosphatase

The HPr kinase of Gram-positive bacteria is an ATP-dependent serine protein kinase, which phosphorylates the HPr protein of the bacterial phosphotrans ferase system (PTS) and is involved in the regulation of carbohydrate metab olism. The hprK gene from Enterococcus faecalis was cloned via polymerase c hain reaction (PCR) and sequenced. The deduced amino acid sequence was conf irmed by microscale Edman degradation and mass spectrometry combined with c ollision-induced dissociation of tryptic peptides derived from the HPr kina se of E. faecalis. The gene was overexpressed in Escherichia coli, which do es not contain any ATP-dependent HPr kinase or phosphatase activity. The ho mogeneous recombinant protein exhibits the expected HPr kinase activity as well as a P-Ser-HPr phosphatase activity, which was assumed to be a separat e enzyme activity. The bifunctional HPr kinase/phosphatase acts preferentia lly as a kinase at high ATP levels of 2 mM occurring in glucose-metabolizin g Streptococci. At low ATP levels, the enzyme hydrolyses P-Ser-HPr. In addi tion, high concentrations of phosphate present under starvation conditions inhibit the HPr kinase activity. Thus, a putative function of the enzyme ma y be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic st ate of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates sugar uptake via the phosphotransferase system (PTS). Reinvestiga tion of the previously described Bacillus subtilis HPr kinase revealed that it also possesses P-Ser-HPr phosphatase activity. However, contrary to the E. faecalis enzyme, ATP alone was not sufficient to switch the phosphatase activity of the B. subtilis enzyme to the kinase activity. A change in act ivity of the B. subtilis HPr kinase was only observed when fructose-1,6-bis phosphate was also present.