Role of a conserved histidine residue, His-195, in the activities of the Escherichia coli mannitol permease.
The mannitol permease, an enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, carries out the transport and phosphorylation of D-mannitol in this organism. Previous studies have shown that His-554 and Cys-384 in the mannitol permease are sequentially phosphorylated in reactions necessary for the transport and phosphorylation of the substrate. These residues are located in a large cytoplasmic domain of the protein. Interaction of the permease with mannitol, and its membrane translocation, however, must involve the N-terminal, transmembrane domain (EIIC domain) of the protein. In this report, we use site-directed mutagenesis and mutant complementation to investigate the role of His-195 in the EIIC domain of the mannitol permease, a residue that is conserved in many PTS permeases. In a previous report [Weng, Q.-P., Elder, J., & Jacobson, G. R. (1992) J. Biol. Chem. 267, 19529-19535], we inferred a role for His-195 that involves its hydrogen-bonding ability. Here we show that His-195 plays a role in high-affinity mannitol binding. Moreover, mutant complementation studies show that a functional His-195 must be on the same subunit as a functional Cys-384 in a permease dimer for phosphotransfer to mannitol to occur. These results and kinetic studies of His-195 mutant proteins imply that His-195 also may play an important role in this phosphotransfer reaction. His-195 is predicted to be in a cytoplasmic "loop" in the EIIC domain of the mannitol permease, in which several other residues have been shown to have roles in mannitol permease activity.
Weng
Q P
QP
Department of Biology, Boston University, Massachusetts 02215.
Jacobson
G R
GR
D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014169:Transformation, Bacterial
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D008353:Mannitol; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli