Mechanism of phosphoryl transfer in the dimeric IIABMan subunit of the Escherichia coli mannose transporter.
The mannose transporter of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) mediates uptake of mannose, glucose, and related hexoses by a mechanism that couples translocation with phosphorylation of the substrate. It consists of the transmembrane IICMan.IIDMan complex and the cytoplasmic IIABMan subunit. IIABMan has two domains (IIA and IIB) that are linked by a 60-A long alanine-proline-rich linker. IIABMan transfers 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. IIABMan occurs as a stable homodimer. The subunit contact is mediated by a swap of beta-strands and an extensive contact area between the IIA domains. The H10C and H175C single and the H10C/H175C double mutants were used to characterize the phosphoryl transfer between IIA to IIB. Subunits do not exchange between dimers under physiological conditions, but slow phosphoryl transfer can take place between subunits from different dimers. Heterodimers of different subunits were produced in vitro by GuHCl-induced unfolding and refolding of mixtures of two different homodimers. With respect to wild-type homodimers, the heterodimers have the following activities: wild-type.H10C, 50%; wild-type.H175C 45%; H10C.H175C, 37%; and wild-type.H10C/H175C (double mutant), 29%. Taken together, this indicates that both cis and trans pathways contribute to the maximal phosphotransferase activity of IIABMan. A phosphoryl group on a IIA domain 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.
Gutknecht
R
R
Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012, Bern, Switzerland.
Flükiger
K
K
Lanz
R
R
Erni
B
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D009154:Mutation; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C037992:phosphoenolpyruvate-mannose phosphotransferase