The
HPr kinase of Gram-positive bacteria is an
ATP-dependent
serine protein kinase, which phosphorylates the
HPr protein of the bacterial
phosphotransferase system (PTS) and is involved in the regulation of carbohydrate metabolism. The hprK gene from Enterococcus faecalis was cloned via polymerase chain reaction (PCR) and sequenced. The deduced amino acid sequence was confirmed by microscale Edman degradation and mass spectrometry combined with collision-induced dissociation of tryptic
peptides derived from the
HPr kinase of E. faecalis. The gene was overexpressed in Escherichia coli, which does not contain any
ATP-dependent
HPr kinase or
phosphatase activity. The homogeneous
recombinant protein exhibits the expected
HPr kinase activity as well as a P-Ser-HPr
phosphatase activity, which was assumed to be a separate
enzyme activity. The bifunctional
HPr kinase/
phosphatase acts preferentially as a
kinase at high
ATP levels of 2 mM occurring in
glucose-metabolizing Streptococci. At low
ATP levels, the
enzyme hydrolyses P-Ser-HPr. In addition, high concentrations of
phosphate present under
starvation conditions inhibit the
HPr kinase activity. Thus, a putative function of the
enzyme may be to adjust the ratio of HPr and P-Ser-HPr according to the metabolic state of the cell; P-Ser-HPr is involved in carbon catabolite repression and regulates
sugar uptake via the
phosphotransferase system (PTS). Reinvestigation 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 activity of the B. subtilis
HPr kinase was only observed when fructose-1,6-bisphosphate was also present.