raffinose and thiomethylgalactoside

To improve the structural organization model of melibiose permease, we assessed the individual contributions of the N-terminal tryptophans to the transporter fluorescence variations induced by the binding of cations and beta-configured sugars, by replacement of the six N-terminal tryptophans by phenylalanines and the study of the signal changes. Only two mutations, W116F located in helix IV and W128F located in the cytoplasmic loop 4-5, impair permease activity. The intrinsic fluorescence spectroscopy analysis of the other mutants suggests that W54, located in helix II, W116, and W128 are mostly responsible for the cation-induced fluorescence variations. These tryptophans, W116 and W128, would also be responsible for the beta-galactoside-induced fluorescence changes observed in the N-terminal domain of the transporter. The implication of W116 and W128 in both the cation- and beta-galactoside-induced fluorescence variations led us to investigate in detail the effects of their mutations on the functional properties of the permease. The results obtained suggest that the domains harboring the two tryptophans, or the residues themselves, play a critical role in the mechanism of Na(+)/sugar symport. Taken together, the results presented in this paper and previous results are consistent with a fundamental role of helix IV in connecting cation- and sugar-binding sites of the melibiose permease.

Topics: Amino Acid Substitution; Binding Sites; Biological Transport; Carbohydrate Metabolism; Carbohydrates; Cations, Monovalent; Escherichia coli; Kinetics; Melibiose; Membrane Transport Proteins; Methylgalactosides; Models, Molecular; Mutation; Nitrophenylgalactosides; Osmolar Concentration; Protein Structure, Secondary; Protein Structure, Tertiary; Proteolipids; Raffinose; Sodium; Spectrometry, Fluorescence; Substrate Specificity; Symporters; Thiogalactosides; Tryptophan; Tyrosine

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.

Topics: alpha-Galactosidase; Biological Transport; Enzyme Induction; Galactose; Lactobacillus; Lactose; Melibiose; Methylgalactosides; Mutation; Nitrophenylgalactosides; Raffinose; Temperature; Thiogalactosides; Time Factors

A Lac+ mutant of Erwinia chrysanthemi was isolated from the Lac- wild type on lactose agar. beta-Galactosidase was expressed independently of lactose transport in both the mutant and the wild type, and neither strain expressed thiogalactoside transacetylase. Lactose transport and alpha-galactosidase, constitutive in the Lac+ strain, were coordinately induced in the Lac- strain by melibiose and raffinose but not by isopropyl-beta-D-thiogalactopyranoside or thiomethyl-beta-D-galactopyranoside. Melibiose was a strong inhibitor of both the melibiose- and the raffinose-induced lactose permeases, whereas raffinose was a strong inhibitor of only the raffinose-induced lactose permease.

Topics: Acetyltransferases; alpha-Galactosidase; beta-Galactosidase; Biological Transport; Disaccharides; Enzyme Induction; Erwinia; Isopropyl Thiogalactoside; Lactose; Melibiose; Methylgalactosides; Mutation; Raffinose; Thiogalactosides