Mechanism of phosphoryl transfer in the dimeric IIAB(Man) subunit of the Escherichia coli mannose transporter

The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotrans ferase system (PTS) mediates uptake of mannose, glucose, and related hexose s by a mechanism that couples translocation with phosphorylation of the sub strate. It consists of the transmembrane IICMan IIDMan complex and the cyto plasmic ILAB(Man) subunit. IIAB(Man) has two domains (IIA and IIB) that are linked by a 60-Angstrom long alanine-proline-rich linker. ILAB(Man) transf ers phosphoryl groups from the phospho-histidine-containing phospho-carrier protein of the PTS to His-10 on IIA, hence to His-175 on IIB, and finally to the 6'-OH of the transported hexose. IIAB(Man) occurs as a stable homodi mer. The subunit contact is mediated by a swap of beta-strands and an exten sive contact area between the IIA domains. The H10C and H175C single and th e H10C/H175C double mutants were used to characterize the phosphoryl transf er between IIA to IIB. Subunits do not exchange between dimers under physio logical conditions, but slow phosphoryl transfer can take place between sub units from different dimers. Heterodimers of different subunits were produc ed in vitro by GuHCl-induced unfolding and refolding of mixtures of two dif ferent homodimers. With respect to wild-type homodimers, the heterodimers h ave the following activities: wild-type.H10C, 50%; wild-type.H175C 45%; H10 C.H175C, 37%; and wild-type.H1OC/H175C (double mutant), 29%. Taken together , this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIAB(Man). A phosphoryl group on a IIA doma in can be transferred either to the IIB domain on the same or on the second subunit in the dimer, and interruption of one of the two pathways results in a reduction of the activity to 70-80% of the control.