EXPRESSION, PURIFICATION, AND KINETIC CHARACTERIZATION OF THE MANNITOL TRANSPORT DOMAIN OF THE PHOSPHOENOLPYRUVATE-DEPENDENT MANNITOL PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI - KINETIC EVIDENCE THAT THE ESCHERICHIA-COLI MANNITOL TRANSPORT PROTEIN IS A FUNCTIONAL DIMER

The overexpression of the membrane-bound C domain of the mannitol tran sport protein EII(Mtl) of Escherichia coli has been achieved. This pro tein, IICMtl, consisting of the first 346 amino acids, was purified fr om membrane vesicles and still bound mannitol with a high affinity. Ge l filtration experiments showed that purified IICMtl was a dimer, conf irming that the interaction within the EII(Mtl) dimer occurs between t he membrane bound portions of the protein. IICMtl in combination with a chimeric protein consisting of the membrane-bound EII(Glc) C domain and the cytoplasmic EII(Mtl) BA domain could restore both phosphoenolp yruvate-dependent phosphorylation and mannitol/mannitol-P exchange act ivity. The interaction in this complex was comparable to that of IICMt l with soluble IIBA(Mtl) in as much as there appeared to be no specifi c interaction between IICMtl and the membrane-bound EII(Glc) C domain; the K-m of IICMtl for the chimer was so low that saturation could not be achieved. In contrast, a very high affinity with a K-m of 2 nM was measured between purified IICMtl and purified EII(Mtl). This interact ion was manifested in a IICMtl-dependent stimulation of the EII(Mtl) c atalyzed phosphoenolpyruvate dependent mannitol phosphorylation reacti on and the mannitol/mannitol-P exchange reaction. The high affinity of IICMtl for the wild type enzyme can be explained by the formation of heterodimers consisting of a IICMtl monomer and an EII(Mtl) monomer wh ich interact at the level of the membrane-bound domains. The 2-fold in crease in mannitol phosphorylation activity of the hetero versus homod imer is an indication that the individual subunits in the homodimer ar e functionally coupled and work at only half their maximum rate. It is known that the EII(Mtl) dimer, but not the monomer, catalyzes the man nitol/mannitol-P exchange reaction. Since the heterodimer also catalyz es this reaction, it appears that only one functional B domain per dim er.