The phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius.: Functional and biochemical characterization of IIABLMan and IIABHMan

Previous studies have suggested that the phosphoenolpyruvate:mannose phosphotransferase system of Streptococcus salivarius consists of a nonphosphorylated enzyme II domain that functions in tandem with a separate enzymatic complex called IIIMan. The IIIMan complex is believed to be composed of two protein dimers with molecular masses of approximately 72 kDa. Analysis of these proteins by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate has indicated that one dimer is composed of two 38.9-kDa subunits called IIIHMan, and the other of two 35.2-kDa subunits called IIILMan. This study was undertaken to determine (1) the number and nature of the phosphorylated residue(s) on IIIHMan and IIILMan and the phosphorylation sequence allowing the transfer of the phosphoryl group from HPr(His similar to P) to the mannose:PTS substrates; (2) whether IIIHMan and IIILMan originate from two different genes or result from a posttranslational modification; and (3) whether these two proteins are involved in the phosphorylation of 2-deoxyglucose, a substrate of the phosphoenolpyruvate:mannose phosphotransferase system. We showed that both IIIHMan and IIILMan were phosphorylated on two histidine residues. One phosphate bond was heat-labile (phosphorylation at the N-1 position of the imidazole ring), while the second was heat-resistant (phosphorylation at the N-3 position of the imidazole ring). The sequence of the first phosphorylation site was deduced by comparing the N-terminal amino acid sequence of both forms of IIIMan with IIA domains of the EII-mannose family. The sequences of both forms were identical over the 15 first amino acids, that is, MIGIIIASHGKFAEG. The sequence of the second phosphorylation site was determined for IIILMan as IHGQVATNxTP, Hence, IIIHMan and IILMan are PTS proteins of the IIAB type and should be renamed IIAB(H)(Man) and IIAB(L)(Man). IIAB(H)(Man) and IIAB(L)(Man) had different peptide profiles after digestion with proteases, indicating that these two proteins are encoded by two different genes. In vitro PEP-dependent phosphorylation assays conducted with a spontaneous mutant devoid of both forms of IIAB(Man) suggested that the phosphoenolpyruvate:mannose phosphotransferase system of S. salivarius is composed of an uncharacterized nonphosphorylated membrane component that works in tandem with IIAB(L)(Man). The physiological functions of IIAB(H)(Man) remain unknown.