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Lactose permease mutants which transport (malto)-oligosaccharides
S G Olsen1, K M Greene, R J Brooker
1Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Journal of Bacteriology
|October 1, 1993
Summary
Researchers engineered lactose permease mutants capable of transporting maltotriose and larger sugar molecules. These multiple mutants, with specific amino acid changes, demonstrate altered substrate specificity in sugar transport.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- Lactose permease (LacY) is a well-studied transporter of the bacterial lactose/galactose family.
- Previous studies identified LacY mutants with altered sugar specificities.
Purpose of the Study:
- To investigate the ability of LacY mutants to transport maltotriose, a trisaccharide.
- To characterize LacY mutants with enhanced transport capabilities for larger oligosaccharides.
Main Methods:
- Screening of previously isolated LacY mutants for maltotriose transport.
- Growth assays on maltotriose-containing media (MacConkey and minimal plates).
- Whole-cell transport assays using alpha-nitrophenylglucoside (αNPG) derivatives.
Main Results:
- Six multiple LacY mutants (five double, one triple) showed fermentation-positive growth on maltotriose.
- These mutants possessed combined amino acid substitutions at positions 177, 236, 306, or 322.
- Multiple mutations, but not single mutations, conferred enhanced maltotriose transport.
- Mutants transported longer αNPG molecules, with up to four glucose residues.
Conclusions:
- Specific combinations of amino acid substitutions in LacY can expand its substrate specificity to include maltotriose.
- The transport of larger oligosaccharides suggests alterations in the permease's binding pocket or translocation mechanism.
- These findings provide insights into the structural determinants of sugar transport by LacY.