Characterisation of a developmentally regulated amino acid transporter gene from Leishmania amazonensis

The metabolism of protozoan parasites of the Leishmania genus is strongly based on amino acid consumption, but little is known about amino acid uptake in these organisms. In the present work, we identified a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid transporter that belongs to the amino acid/auxin permease family, a group of H(+)/amino acid symporters. This single copy gene is upregulated in amastigotes, the life cycle stage found in the mammalian host. La-PAT1 putative orthologous sequences were identified in Leishmania infantum, Leishmania donovani, Leishmania major and Trypanosoma.     

Control of H<Superscript>+</Superscript>/Lactose Coupling by Ionic Interactions in the Lactose Permease of<Emphasis Type="Italic">Escherichia coli</Emphasis>

A combinatorial approach was used to study putative interactions among six ionizable residues (Asp-240, Glu-269, Arg-302, Lys-319, His-322, and Glu-325) in the lactose permease. Neutral mutations were made involving five ion pairs that had not been previously studied. Double mutants, R302L/E325Q and D240N/H322Q, had moderate levels of downhill [¹⁴C]-lactose transport. Mutants in which only one of these six residues was left unchanged (pentuple mutants) were also made. A Pent269⁻ mutant (in which only Glu-269 remains) catalyzed a moderate level of downhill lactose transport. Pent240⁻ and Pent 322⁺ also showed low levels of downhill lactose transport. Additionally, a Pent240⁻ mutant exhibited proton transport upon addition of melibiose, but not lactose. This striking result demonstrates that neutralization of up to five residues of the lactose permease does not abolish proton transport. A mutant with neutral replacements at six ionic residues (hextuple mutant) had low levels of downhill lactose transport, but no uphill accumulation or proton transport. Since none of the mutants in this study catalyzes active accumulation of lactose, this is consistent with other reports that have shown that each residue is essential for proper coupling. Nevertheless, none of the six ionizable residues is individually required for substrate-induced proton cotransport. These results suggest that the H⁺ binding domain may be elsewhere in the permease or that cation binding may involve a flexible network of charged residues.This revised version was published online in August 2005 with a corrected cover date.     

Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond-beta-D-glucosidase

Alkyl beta-D-glucosides were synthesized from D-glucose and alcohols by reverse hydrolysis using the commercially available almond beta-D-glucosidase in 9:1 (v/v) acetonitrile-water medium. The main characteristics of this enzyme-catalyzed glucosylation were established by using 2-hydroxybenzyl alcohol. The reaction is entirely regio- and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1-phenylethyl alcohol. Moreover, by using almond beta-D-glucosidase it was also possible to synthesize alkyl beta-D-galactosides. (c) 1995 John Wiley & Sons, Inc.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibacterial activity of phosphono dipeptides based on 1-amino-1-methylethanephosphonic acid

Phosphono dipeptides containing 1-amino-1-methylethanephosphonic acid (phosphonic acid analogue of alpha-methylalanine, MeAlaP) and glycine, alanine, valine, leucine phenylalanine, proline, methionine or lysine as N- terminal component were synthesized in order to determine their antibacterial properties. Peptides containing alanine, leucine, valine phenylalanine and methionine showed marked in vitro activity, especially against Escherichia coli and Serratia marcescens strains. There were, however, generally less potent than the respective phosphono dipeptides based on 1-aminoethanephosphonic acid (phosphonic acid analogue of alanine, AlaP). The possible mechanism of action of the peptides of MeAlaP involves their active transport into the bacterial cell, followed by intracellular release of MeAlaP, which most likely inhibits alanine racemase, a key enzyme in peptidoglycan biosynthesis. Studies on the uptake of AlaMeAlaP and LeuMeAlaP by Escherichia coli mutants defective in the oligopeptide permease suggest that these peptides are not transported by the oligopeptide transport system.     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Nitrogen regulation of transport operons: analysis of promoters argTr and dhuA

Summary In Salmonella typhimurium the periplasmic permeases for histidine and for lysine-arginine-ornithine are regulated by nitrogen availability. The nature of the dhuA and argTr promoters of the operons coding for these permeases was analyzed by placing the galactokinase gene under their control (in vector pKO-1). argTr was found to respond to nitrogen regulation. We investigated the involvement of a mirror symmetry in argTr in its regulation by nitrogen. It had been postulated previously (Higgins and Ames 1982) that mirror symmetries might act as protein recognition sites important in regulation of gene expression. Here we demonstrate that the mirror symmetry in argTr is not involved in nitrogen control. Contrary to expectation, the galK gene was not regulated by nitrogen when it was placed under dhuA control. Here we propose a possible explanation for this finding.     

Structural characterization of the osmosensor ProP

ProP, an osmoprotectant symporter from the major facilitator superfamily was expressed, purified and reconstituted into proteoliposomes that are amenable to structural characterization using infrared spectroscopy. Infrared spectra recorded in both ¹H₂O and ²H₂O buffers reveal amide I band shapes that are characteristic of a predominantly α-helical protein, and that are similar to those recorded from the well-characterized homolog, lactose permease (LacY). Curve-fit analysis shows that ProP and LacY both exhibit a high α-helical content. Both proteins undergo extensive peptide hydrogen-deuterium exchange after exposure to ²H₂O, but are surprisingly thermally stable with denaturation temperatures greater than 60 °C. 25-30% of the peptide hydrogens in both ProP and LacY are resistant to exchange after 72 h in ²H₂O at 4 °C. Surprisingly, these exchange resistant peptide hydrogens exchange completely for deuterium at temperatures below those that lead to denaturation. Our results show that ProP adopts a highly α-helical fold similar to that of LacY, and that both transmembrane folds exhibit unusually high temperature-sensitive solvent accessibility. The results provide direct evidence that ProP adopts a structure consistent with other major facilitator superfamily members.