Cytoplasmic phosphorylating domain of the mannitol-specific transport protein of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli: overexpression, purification, and functional complementation with the mannitol binding domain.
The cytoplasmic C-terminal domain, residues 348-637, and the membrane-bound N-terminal domain, residues 1-347, of EIImtl have been subcloned and expressed in Escherichia coli. The N-terminal domain, IICmtl, contains the mannitol binding site, and the C-terminal domain, IIBAmtl, contains the activity-linked phosphorylation sites, His-554 and Cys-384. Overexpression of the BA domain was achieved by a translational in-frame fusion of the gene with the cro ATG start codon, downstream of the strong PR promoter of phage lambda. The domain has been purified and characterized in in vitro complementation assays. It possessed no mannitol phosphorylation activity itself but was able to restore the phosphoenolpyruvate-dependent phosphorylation activity of two EIImtl phosphorylation site mutants, lacking His-554 or Cys-384. The complementary N-terminal domain was also expressed. Membranes possessing IICmtl were unable to phosphorylate mannitol at the expense of phosphoenolpyruvate. However, when the membranes were combined with the purified C-terminal domain, mannitol phosphorylation activity was restored. Mannitol transport and phosphorylation were also restored in vivo when the two plasmids encoding the N- and C-terminal domains were expressed in the same cell. These data demonstrate the existence of structurally and functionally distinct domains in EIImtl: a cytoplasmic domain with phosphorylating activity and a membrane-bound N-terminal domain which, in the presence of the cytoplasmic domain, is able to actively transport and phosphorylate mannitol. The ability to separate, overproduce, and purify structurally stable, enzymatically active domains opens the way for 3D structural studies as well as complete kinetic analysis of the activities of the individual domains and their interactions.
van Weeghel
R P
RP
BISON Research Institute, University of Groningen, The Netherlands.
Meyer
G
G
Pas
H H
HH
Keck
W
W
Robillard
G T
GT
D001483:Base Sequence; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides / Q000737:chemistry; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry* / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D013329:Structure-Activity Relationship
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D009841:Oligonucleotides; D010750:Phosphoproteins; D008353:Mannitol; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C022358:mannitol PTS permease, E coli