EXPRESSION, PURIFICATION AND CHARACTERIZATION OF THE ENZYME-II MANNITOL-SPECIFIC DOMAIN FROM STAPHYLOCOCCUS-CARNOSUS AND DETERMINATION OF THE ACTIVE-SITE CYSTEINE RESIDUE

The C-terminal B domain of mannitol-specific enzyme II (enzyme IIB) of the phosphoenolpyruvate-dependent phosphotransferase system for manni tol from Sml,hylococclls carnosus was subcloned, purified and characte rized, In Staphylococcal cells, mannitol-specific enzyme II is compose d of a soluble A domain (EIIA) and a transmembrane C domain transporte r with a fused enzyme II B (IIB) domain. We purified large amounts of the IIB domain as an in-frame fusion with six histidine residues. Here , we show that the domain is stable and can be phosphorylated by phosp hoenolpyruvate and the phosphotransferase components. It is a dimer ov er a wide range of pH values and salt conditions. Differences between the published nucleotide sequence data and the mass-spectroscopic data obtained with the purified protein lead to anewed nucleotide sequenci ng of the gene. Two errors in the original proposed sequence were foun d, the correction of the second error leading to a frame shift that ad ds 10 amino acids to the deduced amino acid sequence. The mass of the phosphorylated domain is 20068 Da, 80 Da more than the mass of the unp hosphorylated domain, therefore, no other residues, such as COOH side chains, are directly involved in an additional phosphate linkage conce rning the IIB domain. P-31-NMR experiments as well as chemical modific ation proved that Cys429 is the phosphoamino acid. Titration of the ph osphorylated domain during P-31-NMR did not lead to the typical shift for the protonation of the thiophosphate in the resonance spectrum. Th us, the thiophosphate remains in the twofold negatively charged state.