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

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Abstract
The mannitol-specific transport protein in Escherichia coli, EIImtl, 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, IIBmtl, 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 IIBAmtl. The gene fragment coding for IIBmtl 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 IIBmtl/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 EIImtl, C384S, and with the transmembrane domain, IICmtl. 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 IIAmtl yielded an apparent Km of P-IIBmtl for IICmtl of 200 µM and an apparent Vmax of 71 nmol of mtl-P min-1 mg of membrane protein)-1. This Vmax value is comparable to that of wild-type EIImtl measured under the same experimental conditions

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