Studies on plant amylases

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp 389 within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp 389 in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a ⌬ldnt1/⌬ldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K m by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn 175 established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn 175 has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp 389 is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn 175 and Asp 389 lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.

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“…Nucleoside transport measurements in L. donovani promastigotes were performed by the oil-stop method described previously (20 (21).…”

Section: Methodsmentioning

confidence: 99%

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

Arastu‐Kapur¹,

Arendt²,

Purnat

et al. 2005

Journal of Biological Chemistry

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“…The acquired data were interpreted by a model formally derived from MichaelisMenten kinetics [31, 321. The Hanes plot [21] (Fig. 11) yielded an apparent K,,, value of approximately 0.246 pM for staphyloferrin A and 58 nM for ferrioxamine E. The v , , , values were estimated to be 82 pmol mg-' .…”

Section: Biological Activitymentioning

confidence: 99%

“…The uptake rate was calculated and the average of three experiments determined. The data were analysed by a Hanes plot [21].…”

Section: Transport Experimentsmentioning

confidence: 99%

Staphyloferrin A: a structurally new siderophore from staphylococci

Konetschny-Rapp

Jung

Meiwes

et al. 1990

European Journal of Biochemistry

140

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Two ferric ion-binding compounds, designated staphyloferrin A and B, were detected in the culture filtrates of staphylococci grown under iron-deficient conditions. Staphyloferrin A was isolated from cultures of Staphylococcus hyicus DSM 20459. The structural elucidation of this highly hydrophilic, acid-labile compound revealed a novel siderophore, N 2 , N 5 -di-(l-oxo-3-hydroxy-3,4-dicarboxybutyl)-~-ornithine, which consists of one ornithine and two citric acid residues linked by two amide bonds. The two citric acid components of staphyloferrin A provide two tridentate pendant ligands, comprising of a /I-hydroxy, P-carboxy-substituted carboxylic acid derivative, for octahedral metal chelation. The CD spectrum of the staphyloferrin A ferric complex indicates a predominant A configuration about the ferric ion center. The uptake of ferric staphyloferrin A by S. hyicus obeys MichaelisMenten kinetics ( K , = 0.246 pM; v,,, = 82 pmol . mg-. min-I), indicating active transport of this siderophore.The staphyloferrin A transport system is different from that of the ferrioxamines as shown by an antagonism test. Production of staphyloferrin A is strongly iron-dependent and is stimulated by supplementation of the medium with either D-or L-ornithine. ~~-[S-'~C]ornithine was incorporated into staphyloferrin A, demonstrating that ornithine is an intermediate in staphyloferrin A biosynthesis.Iron, the fourth most abundant element of the earths crust, is required by all aerobic microorganisms and plays an important role in membrane-bound electron-transport chains as well as in cytoplasmic redox enzymes. In an aerobic environment iron is present in the Fe(II1) oxidized form, which polymerizes into insoluble ferric oxyhydroxides under physiological conditions [l, 21. In mammals, iron is bound to proteins such as lactoferrin and transferrin, and is therefore inaccessible, by direct means, to microorganisms [3]. Thus, microorganisms have developed different strategies to provide themselves with iron. The most common and well-investigated strategy is the high-affinity Fe( 111) uptake via siderophores. During iron deficiency, microorganisms excrete low-molecular-mass iron(II1)-chelating compounds (siderophores) in their iron-free form. After ferric ion chelation, the complexes are actively taken up by specific transport systems [4]. Several hundred siderophores from prokaryotes and eukaryotes are now known, which can structurally be assigned to the major classes of hydroxamate-and catecholate-type siderophores.

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“…2) and followed Michaelis-Menten kinetics. The K , value (Hanes, 1932) was 60 k 2 PM and the maximal velocity (Vmax) was 29 & 1 nmol min-l (mg protein)-'. Control experiments with solid NaCl confirmed these results.…”

Section: Active Glycine Betaine Transport and Its Kineticsmentioning

confidence: 99%

Transport of glycine betaine in the extremely haloalkaliphilic sulphur bacterium Ectothiorhodospira halochloris

Peters¹,

Tel‐Or²,

Trüper³

1992

Journal of General Microbiology

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The effect of osmotic stress on the transport of the compatible solute glycine betaine was examined in Ectothiorhodospira halochforis, an extremely haloalkaliphilic, phototrophic sulphur bacterium. Kinetic data indicated that E. halochforis possesses an active transport system for glycine betaine which is saturable and exhibits Michaelis-Menten kinetics. Experiments with chloramphenicol-treated cells (50 pg per ml of cell suspension) indicated that the transport system is constitutive and might be activated by a change in osmotic pressure. The uncouplers carbonyl cyanide rn-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (CCFP) totally blocked uptake at concentrations of 25 p~ and 100 p~, respectively. The system was insensitive to the cytochrome oxidase inhibitor sodium azide (1 mM), the respiratory chain inhibitor potassium cyanide (1 mM) and the glycolysis inhibitor iodoacetate (1 mM). The ionophore nigericin (50 p~) had the greatest inhibitory effect, completely abolishing uptake, while monensin (100 p~) caused 80% inhibition. Glycine betaine transport possessed considerable structural specifity: proline betaine was the most effective competitor and trigonelline and dimethylglycine exerted inhibition to a lesser extent. Transport in the dark was at a greatly reduced rate. These results collectively implied that the specific transport of glycine betaine might be driven by the electrochemical-proton gradient generated by anaerobic photosynthesis.

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Studies on plant amylases

Cited by 686 publications

References 3 publications

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

Staphyloferrin A: a structurally new siderophore from staphylococci

Transport of glycine betaine in the extremely haloalkaliphilic sulphur bacterium Ectothiorhodospira halochloris

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