EXPRESSION AND CHARACTERIZATION OF A STRUCTURAL AND FUNCTIONAL DOMAIN OF THE MANNITOL-SPECIFIC TRANSPORT PROTEIN INVOLVED IN THE COUPLING OF MANNITOL TRANSPORT AND PHOSPHORYLATION IN THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI

The mannitol-specific transport protein in Escherichia coli, EII(mtl), consists of three structural and functional domains: a hydrophilic EIII-like domain (the A domain); a hydrophobic transmembrane domain (the C domain); and a second hydrophilic domain (the B domain) which connects the A and C domains together. The A domain contains the first phosphorylation site, His554, while the B domain contains the second phosphorylation site, Cys384. The phosphoryl group which is needed for the active transport of mannitol is sequentially transferred from P-enolpyruvate via the two phosphorylation sites to mannitol bound to the substrate binding site. In this paper, the expression, purification, and initial characterization of the B domain, IIB(mtl), are described. Oligonucleotide-directed mutagenesis was used to produce an amber stop codon (TAG) and HindIII restriction site in a flexible loop between the B and A domains in the subcloned gene fragment coding for IIBA(mtl) (van Weeghel et al., 1991c). The gene fragment coding for IIB(mtl) was then subcloned behind strong promoters, located in two different expression/mutagenesis vectors, which directed the expression of the 15.3-kDa polypeptide in Escherichia coli. The domain was purified from E. coli crude cell extracts by using Q-Sepharose Fast Flow, S-Sepharose Fast Flow, and hydroxylapatite column steps. This purification procedure resulted in 1 mg of pure IIB(mtl)/g of cell, wet weight. The purified B domain was analyzed in vitro for its catalytic activity with membranes containing the phosphorylation site mutant form of EII(mtl) C384S, and with the transmembrane domain, IIC(mtl). The B domain, together with purified IIA, was able to restore the P-enolpyruvate-dependent phosphorylation activity of the membrane-bound C domain. Steady-state mannitol phosphorylation kinetics at saturating EI, HPr, and IIA(mtl) yielded an apparent K(m) of P-IIB(mtl) for IIC(mtl) of 200 muM and an apparent V(max) of 71 nmol of mtl-P min-1 mg of membrane protein)-1. This V(max) value is comparable to that of wild-type EII(mtl) measured under the same experimental conditions.