Changing the lactose permease of Escherichia coli into a galactose-specific symporter.

Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print Cited Medium: Print ISSN: 0027-8424 (Print) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE

Original Publication: Washington, DC : National Academy of Sciences

Calcium-Binding Proteins* ; Escherichia coli Proteins* ; Periplasmic Binding Proteins*; Escherichia coli/*enzymology ; Galactose/*metabolism ; Membrane Transport Proteins/*metabolism ; Monosaccharide Transport Proteins/*metabolism ; Symporters/*metabolism; Alanine/genetics ; Alanine/metabolism ; Cysteine/genetics ; Cysteine/metabolism ; Disaccharides/metabolism ; Enzyme Inhibitors/pharmacology ; Escherichia coli/genetics ; Ethylmaleimide/pharmacology ; Lactose/metabolism ; Membrane Transport Proteins/genetics ; Mutagenesis, Site-Directed ; Substrate Specificity

N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and beta,d-galactopyranosyl 1-thio-beta,D-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [(14)C]NEM. Remarkably, however, D-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [(14)C]galactose transport by lactose, melibiose, or beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 --> Ser causes only mild inhibition of lactose transport, the mutations Ala-122 --> Phe and Ala-122 --> Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the non-galactosyl moiety of D-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of D-galactopyranosides.

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R01 DK051131 United States DK NIDDK NIH HHS; R56 DK051131 United States DK NIDDK NIH HHS; DK51131:06 United States DK NIDDK NIH HHS

0 (Calcium-Binding Proteins)
0 (Disaccharides)
0 (Enzyme Inhibitors)
0 (Escherichia coli Proteins)
0 (LacY protein, E coli)
0 (Membrane Transport Proteins)
0 (Monosaccharide Transport Proteins)
0 (Periplasmic Binding Proteins)
0 (Symporters)
0 (galactose-binding protein)
9068-45-5 (lactose permease)
J2B2A4N98G (Lactose)
K848JZ4886 (Cysteine)
O3C74ACM9V (Ethylmaleimide)
OF5P57N2ZX (Alanine)
X2RN3Q8DNE (Galactose)

Date Created: 20020516 Date Completed: 20020618 Latest Revision: 20181113

20221213

PMC124451

10.1073/pnas.102178299

12011425