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Histidine-containing protein (HPr) of gram-positive bacteria was found to be phosphorylated at a seryl residue (P-ser-HPr) in an ATP-dependent reaction catalyzed by a protein kinase (J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. U.S.A. 80:6790-6794, 1983). Here we describe the purification and characterization of a soluble enzyme of Streptococcus faecalis which splits the phosphoryl bond in P-ser-HPr. The enzyme has a molecular weight of ca. 7.5 x 104, as determined by its migration behavior on a Sephacryl S-200 column. On native polyacrylamide gels the purified enzyme produced only one protein band. On sodium dodecyl sulfate-polyacrylamide gels we found one major protein band of molecular weight 2.9 x 104 and two minor protein bands of molecular weights 2.3 x 104 and 7 x 104. Fructose 1,6-diphosphate, which stimulated the ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, had no effect on the phosphatase activity. Other glycolytic intermediates also had no effect. However, inorganic phosphate, which inhibited the ATP-dependent HPr kinase, stimulated the P-ser-HPr phosphatase. EDTA at a concentration of 0.1 mM completely inhibited the phosphatase. Divalent cations like Mg2+, Mn2+, and Co2, overcame the inhibition by EDTA. Fe2+, Zn2+, and Cu2+ had no effect, whereas Ca2+ slightly inhibited the phosphatase. ATP was also found to inhibit the phosphatase. Under conditions in which ATP severely inhibited the phosphatase, ADP was found to have no effect on the enzyme activity. Besides P-ser-HPr of S. faecalis, the phosphatase was also able to hydrolyze the phosphoryl bond in P-ser-HPr of Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis,

The histidine-containing protein (HPr) of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) was isolated from Staphylococcus carnosus and purified to homogeneity. In Gram-positive Staphylococci and Streptococci the phosphoenolpyruvate-dependent bacterial phosphotransferase system (PTS) represents the main sugar-transport system, catalyzing the vectorial phosphorylation of sugar during transport into the cytoplasm. In these bacteria most sugars are transported by this multienzyme complex, consisting of the two general, constitutively expressed cytoplasmic proteins enzyme I and histidine-containing protein (HPr) and different sugar-specific, inducible and membrane-bound enzyme II/enzyme 111 complexes. In an initial step phosphoenolpyruvate phosphorylates the protein kinase enzyme I in a Mg2+-dependent reaction at the N' atom of a histidine residue. From the phosphorylated enzyme I the phospho group is transferred to the catalytic site of the central phosphocarrier HPr to the N" atom of Hisl5. HPr phosphorylated at His15 serves as a phospho group distributor by phosphorylating the different sugar-specific enzyme III/enzyme I1 complexes. Finally, the phosphorylated enzyme I1 catalyzes the membrane translocation of a sugar molecule under concomitant phosphorylation. For a detailed overview, the reader is referred to one of the recent reviews [l].In contrast to Gram-negative bacteria, it was found that the sugar uptake in Gram-positive bacteria via PTS is regulated by an ATP-dependent HPr kinase [2], phosphorylating Correspondence to W. Hengstenberg,