Back to Search
Start Over
Domain complementation studies reveal residues critical for the activity of the mannitol permease from Escherichia coli.
- Source :
-
Biochimica et biophysica acta [Biochim Biophys Acta] 2009 Feb; Vol. 1788 (2), pp. 581-6. Date of Electronic Publication: 2008 Oct 29. - Publication Year :
- 2009
-
Abstract
- This paper presents domain complementation studies in the mannitol transporter, EIImtl, from Escherichia coli. EIImtl is responsible for the transport and concomitant phosphorylation of mannitol over the cytoplasmic membrane. By using tryptophan-less EIImtl as a basis, each of the four phenylalanines located in the cytoplasmic loop between putative transmembrane helices II and III in the membrane-embedded C domain were replaced by tryptophan, yielding the mutants W97, W114, W126, and W133. Except for W97, these single-tryptophan mutants exhibited a high, wild-type-like, binding affinity for mannitol. Of the four mutants, only W114 showed a high mannitol phosphorylation activity. EIImtl is functional as a dimer and the effect of these mutations on the oligomeric activity was investigated via heterodimer formation (C/C domain complementation studies). The low phosphorylation activities of W126 and W133 could be increased 7-28 fold by forming heterodimers with either the C domain of W97 (IICmtlW97) or the inactive EIImtl mutant G196D. W126 and W133, on the other hand, did not complement each other. This study points towards a role of positions 97, 126 and 133 in the oligomeric activation of EIImtl. The involvement of specific residue positions in the oligomeric functioning of a sugar-translocating EII protein has not been presented before.
- Subjects :
- Amino Acid Sequence
Enzyme Activation
Escherichia coli genetics
Escherichia coli Proteins genetics
Mannitol metabolism
Molecular Sequence Data
Monosaccharide Transport Proteins genetics
Mutation genetics
Phosphoenolpyruvate Sugar Phosphotransferase System genetics
Phosphorylation
Protein Multimerization
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Escherichia coli enzymology
Escherichia coli Proteins chemistry
Escherichia coli Proteins metabolism
Monosaccharide Transport Proteins chemistry
Monosaccharide Transport Proteins metabolism
Phosphoenolpyruvate Sugar Phosphotransferase System chemistry
Phosphoenolpyruvate Sugar Phosphotransferase System metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-3002
- Volume :
- 1788
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta
- Publication Type :
- Academic Journal
- Accession number :
- 19013424
- Full Text :
- https://doi.org/10.1016/j.bbamem.2008.10.008