EXPRESSION AND CHARACTERIZATION OF A STRUCTURAL AND FUNCTIONAL DOMAINOF 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 EI II-like domain (the A domain); a hydrophobic transmembrane domain (the C domain); and a second hydrophilic domain (the B domain) which conne cts the A and C domains together. The A domain contains the first phos phorylation site, His554, while the B domain contains the second phosp horylation site, Cys384. The phosphoryl group which is needed for the active transport of mannitol is sequentially transferred from P-enolpy ruvate via the two phosphorylation sites to mannitol bound to the subs trate binding site. In this paper, the expression, purification, and i nitial characterization of the B domain, IIB(mtl), are described. Olig onucleotide-directed mutagenesis was used to produce an amber stop cod on (TAG) and HindIII restriction site in a flexible loop between the B and A domains in the subcloned gene fragment coding for IIBA(mtl) (va n Weeghel et al., 1991c). The gene fragment coding for IIB(mtl) was th en subcloned behind strong promoters, located in two different express ion/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 re sulted 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, an d with the transmembrane domain, IIC(mtl). The B domain, together with purified IIA, was able to restore the P-enolpyruvate-dependent phosph orylation activity of the membrane-bound C domain. Steady-state mannit ol phosphorylation kinetics at saturating EI, HPr, and IIA(mtl) yielde d an apparent K(m) of P-IIB(mtl) for IIC(mtl) of 200 muM and an appare nt 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.