Identification of valine 177 as a mutation altering specificity for transport of sugars by the Escherichia coli lactose carrier. Enhanced specificity for sucrose and maltose.
A mutant of the Escherichia coli lactose carrier has been selected (in an invertase-positive strain) based on its ability to grow on 6 mM sucrose in a manner dependent upon lactose carrier induction by isopropyl-1-thio-beta-D-galactopyranoside. The mutant was cloned, and DNA sequencing revealed a point mutation in lacY which changed alanine 177 to valine. The valine 177 mutation increased the transport rate for both [14C]sucrose and the maltose analog 4-nitrophenyl-alpha-maltoside. The potency for inhibition of beta-ONPG transport by several sugars containing the glucopyranosyl moiety (maltose, cellobiose, or palatinose) was increased significantly relative to the parental carrier. Similar experiments showed that the mutation did not affect the affinity for such commonly studied substrates as 4-nitrophenyl-alpha-D-galactopyranoside and beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside. These data indicate that gross structural alteration of the galactoside binding site cannot account for increased transport of sucrose and maltose by the valine 177 mutant. We conclude that effects of the valine 177 mutation are not limited strictly to changes in observed sugar affinity and that sugar-specific changes in turnover number may be an important determinant of the altered spectrum of sugar specificities exhibited by the Val-177 carrier. These phenomena may be related to the effect of this mutation on proton recognition (described in King, S.C., and Wilson, T.H. (1990) J. Biol. Chem. 265, 9645-9651).
King
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SC
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
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TH
D001692:Biological Transport; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D005838:Genotype; D007700:Kinetics; D008320:Maltose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010957:Plasmids; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism*; D027981:Symporters; D014633:Valine
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013395:Sucrose; D008320:Maltose; C021499:lactose permease; D014633:Valine