Expression, purification, and kinetic characterization of the mannitol transport domain of the phosphoenolpyruvate-dependent mannitol phosphotransferase system of Escherichia coli. Kinetic evidence that the E. coli mannitol transport protein is a functional dimer

• Published in: The Journal of biological chemistry Vol. 269; no. 27; pp. 17863 - 17871
• Main Authors: BOER, H, TEN HOEVE-DUURKENS, R. H, SCHUURMAN-WOLTERS, G. K, DIJKSTRA, A, ROBILLARD, G. T
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 08.07.1994
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Biological Transport; Biopolymers; Cloning, Molecular; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Escherichia coli - enzymology; Escherichia coli Proteins; Fundamental and applied biological sciences. Psychology; Kinetics; Mannitol - metabolism; Mass Spectrometry; Molecular Sequence Data; Monosaccharide Transport Proteins; Phosphoenolpyruvate Sugar Phosphotransferase System - chemistry; Phosphoenolpyruvate Sugar Phosphotransferase System - metabolism; Proteins; Purification; C terminal peptide; Enzyme; Escherichia coli; Phosphoenolpyruvate-dependent phosphotransferase system; Mannitol; Bacteria; Carbohydrate; Dimer; Kinetics; Recombinant protein; Carrier protein; Enterobacteriaceae
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)32389-X

The overexpression of the membrane-bound C domain of the mannitol transport protein EIIMtl of Escherichia coli has been achieved. This protein, IICMtl, consisting of the first 346 amino acids, was purified from membrane vesicles and still bound mannitol with a high affinity. Gel filtration experiments showed that purified IICMtl was a dimer, confirming that the interaction within the EIIMtl dimer occurs between the membrane-bound portions of the protein. IICMtl in combination with a chimeric protein consisting of the membrane-bound EIIGlc C domain and the cytoplasmic EIIMtl BA domain could restore both phosphoenolpyruvate-dependent phosphorylation and mannitol/mannitol-P exchange activity. The interaction in this complex was comparable to that of IICMtl with soluble IIBAMtl in as much as there appeared to be no specific interaction between IICMtl and the membrane-bound EIIGlc C domain; the Km of IICMtl for the chimer was so low that saturation could not be achieved. In contrast, a very high affinity with a Km of 2 nM was measured between purified IICMtl and purified EIIMtl. This interaction was manifested in a IICMtl-dependent stimulation of the EIIMtl catalyzed phosphoenolpyruvate-dependent mannitol phosphorylation reaction 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 EIIMtl monomer which interact at the level of the membrane-bound domains. The 2-fold increase in mannitol phosphorylation activity of the hetero- versus homodimer is an indication that the individual subunits in the homodimer are functionally coupled and work at only half their maximum rate. It is known that the EIIMtl dimer, but not the monomer, catalyzes the mannitol/mannitol-P exchange reaction. Since the heterodimer also catalyzes this reaction, it appears that only one functional B domain is required per dimer.