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2.
J Plant Physiol ; 171(12): 993-7, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24974325

ABSTRACT

The mechanism of iron uptake in the chrysophyte microalga Dinobryon was studied. Previous studies have shown that iron is the dominant limiting elements for growth of Dinobryon in the Eshkol reservoir in northern Israel, which control its burst of bloom. It is demonstrated that Dinobryon has a light-stimulated ferrireductase activity, which is sensitive to the photosynthetic electron transport inhibitor DCMU and to the uncoupler CCCP. Iron uptake is also light-dependent, is inhibited by DCMU and by CCCP and also by the ferrous iron chelator BPDS. These results suggest that ferric iron reduction by ferrireductase is involved in iron uptake in Dinobryon and that photosynthesis provides the major reducing power to energize iron acquisition. Iron deprivation does not enhance but rather inhibits iron uptake contrary to observations in other algae.


Subject(s)
Chrysophyta/metabolism , Iron/metabolism , Microalgae/metabolism , Chrysophyta/drug effects , Chrysophyta/growth & development , Chrysophyta/radiation effects , Culture Media/pharmacology , Enzyme Inhibitors/pharmacology , FMN Reductase/antagonists & inhibitors , FMN Reductase/metabolism , Iron/pharmacology , Light , Microalgae/drug effects , Microalgae/growth & development , Microalgae/radiation effects , Phenanthrolines/pharmacology , Photosynthesis/drug effects , Photosynthesis/radiation effects , Time Factors
3.
Biometals ; 23(6): 1029-42, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20508972

ABSTRACT

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.


Subject(s)
Chlorella/enzymology , FMN Reductase/antagonists & inhibitors , Iron Chelating Agents/pharmacology , Cell Membrane/drug effects , Chlorella/drug effects , Deferoxamine/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Hydroxamic Acids/pharmacology , Iron/administration & dosage , Siderophores/pharmacology , Glyphosate
4.
Mol Biochem Parasitol ; 171(1): 17-24, 2010 May.
Article in English | MEDLINE | ID: mdl-20093143

ABSTRACT

Infections with the microaerophilic protozoan parasite Trichomonas vaginalis are commonly treated with metronidazole, a 5-nitroimidazole drug. Metronidazole is selectively toxic to microaerophiles and anaerobes because reduction at the drug's nitro group, which is a precondition for toxicity, occurs only quantitatively in these organisms. In our previous work we identified the flavin enzyme thioredoxin reductase as an electron donor to 5-nitroimidazole drugs in T. vaginalis and observed that highly metronidazole-resistant cell lines lack thioredoxin reductase and flavin reductase activities. In this study we added the flavin inhibitor diphenyleneiodonium (DPI) to T. vaginalis cultures in order to test our hypothesis that metronidazole reduction is catalyzed by flavin enzymes, e.g. thioredoxin reductase, and intracellular free flavins. Indeed, within hours, DPI rendered T. vaginalis insensitive to metronidazole concentrations as high as 1mM and prevented the formation of metronidazole adducts with proteins. Thioredoxin reductase activity was absent from DPI-treated cells and flavin reductase activity was sharply decreased. In addition, DPI-treated cells also upregulated the expression of antioxidant enzymes, i.e. thioredoxin peroxidases and superoxide dismutases, and displayed a fundamentally altered metabolism caused by inactivation of pyruvate:ferredoxin oxidoreductase (PFOR) and concomitant upregulation of lactate dehydrogenase (LDH) activity. Thus, the disruption of the cellular flavin metabolism by DPI mediated metabolic steps which are similar to that of cells with metronidazole resistance induced in vitro. Finally, we present direct evidence that the increased expression of antioxidant enzymes is dispensable for acquiring resistance to metronidazole.


Subject(s)
Enzyme Inhibitors/pharmacology , FMN Reductase/antagonists & inhibitors , Flavins/antagonists & inhibitors , Metronidazole/pharmacology , Onium Compounds/pharmacology , Thioredoxin-Disulfide Reductase/antagonists & inhibitors , Trichomonas vaginalis/drug effects , Antiprotozoal Agents/metabolism , Antiprotozoal Agents/pharmacology , Drug Resistance , Metronidazole/metabolism , Prodrugs/metabolism , Prodrugs/pharmacology
5.
J Agric Food Chem ; 57(20): 9569-74, 2009 Oct 28.
Article in English | MEDLINE | ID: mdl-19780538

ABSTRACT

Previous research demonstrated that plant nutrient assimilation was reduced by glyphosate (Gly). A 2 year field experiment investigated the effects of Gly at drift rate (12.5% of commercial use rate) on Fe concentrations in leaves and seeds of Gly-sensitive (GS) soybean, and a greenhouse experiment evaluated Gly effects on Fe assimilation using root in vivo ferric reductase activity (FRA) in two GS and one Gly-resistant (GR) soybean cultivars. Field studies showed that Gly drift rates resulted in a significant decrease in the Fe concentration in seeds and leaves compared to the nontreated plants. In greenhouse studies, leaf Fe and FRA were inhibited in GS cultivars Hutcheson and DP 5110 and the GR cultivar AG 4604RR and leaf Fe was positively correlated with root FRA (p < 0.0001). These results indicate that Gly can interfere with Fe assimilation in both GS and GR soybean. Understanding the implication of Gly on Fe nutrition in soybean seed would help soybean agronomists and breeders seeking to improve seed mineral nutrition qualities.


Subject(s)
FMN Reductase/metabolism , Glycine max/drug effects , Glycine/analogs & derivatives , Herbicides/pharmacology , Iron/metabolism , Plant Proteins/metabolism , Plant Roots/enzymology , Seeds/metabolism , FMN Reductase/antagonists & inhibitors , Glycine/pharmacology , Plant Proteins/antagonists & inhibitors , Plant Roots/drug effects , Seeds/drug effects , Glycine max/enzymology , Glycine max/metabolism , Glyphosate
6.
Arch Biochem Biophys ; 483(1): 29-36, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19138657

ABSTRACT

The ferric reductase B (FerB) protein of Paracoccus denitrificans exhibits activity of an NAD(P)H: Fe(III) chelate, chromate and quinone oxidoreductase. Sequence analysis places FerB in a family of soluble flavin-containing quinone reductases. The enzyme reduces a range of quinone substrates, including derivatives of 1,4-benzoquinone and 1,2- and 1,4-naphthoquinone, via a ping-pong kinetic mechanism. Dicoumarol and Cibacron Blue 3GA are competitive inhibitors of NADH oxidation. In the case of benzoquinones, FerB apparently acts through a two-electron transfer process, whereas in the case of naphthoquinones, one-electron reduction takes place resulting in the formation of semiquinone radicals. A ferB mutant strain exhibited an increased resistance to 1,4-naphthoquinone, attributable to the absence of the FerB-mediated redox cycling. The ferB promoter displayed a high basal activity throughout the growth of P. denitrificans, which could not be further enhanced by addition of different types of naphthoquinones. This indicates that the ferB gene is expressed constitutively.


Subject(s)
FMN Reductase/chemistry , FMN Reductase/metabolism , NAD(P)H Dehydrogenase (Quinone)/chemistry , NAD(P)H Dehydrogenase (Quinone)/metabolism , Paracoccus denitrificans/enzymology , Amino Acid Sequence , Base Sequence , DNA, Bacterial/genetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , FMN Reductase/antagonists & inhibitors , FMN Reductase/genetics , Genes, Bacterial , Kinetics , Molecular Sequence Data , Mutation , NAD(P)H Dehydrogenase (Quinone)/antagonists & inhibitors , NAD(P)H Dehydrogenase (Quinone)/genetics , Paracoccus denitrificans/genetics , Promoter Regions, Genetic , Quinones/metabolism , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Substrate Specificity
7.
New Phytol ; 177(4): 899-906, 2008.
Article in English | MEDLINE | ID: mdl-18179601

ABSTRACT

Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.


Subject(s)
FMN Reductase/antagonists & inhibitors , Glycine/analogs & derivatives , Helianthus/drug effects , Iron Deficiencies , Plant Roots/drug effects , Plant Roots/enzymology , Glycine/pharmacology , Helianthus/metabolism , Herbicides/pharmacology , Plant Leaves/drug effects , Plant Leaves/metabolism , Time Factors , Glyphosate
9.
Free Radic Biol Med ; 37(5): 713-23, 2004 Sep 01.
Article in English | MEDLINE | ID: mdl-15288128

ABSTRACT

Nitric oxide (NO) has been found to inhibit the actions of the transmembrane metal reductase Fre1 in the yeast Saccharomyces cerevisiae. This membrane-spanning heme protein is homologous to the gp91(PHOX) protein of the NADPH oxidase enzyme complex and is responsible for reducing extracellular oxidized metals (i.e., ferric and cupric ions) before high-affinity uptake. Consistent with its role in metal metabolism, inhibition of Fre1 by NO also inhibited yeast growth in low-iron medium. Inhibition by NO was found to be O(2)-dependent and irreversible. Further examination of the chemistry responsible for activity loss shows that the generation of N(2)O(3) via NO-O(2) chemistry was responsible for the activity loss, possibly via nitrosation of the protein followed by loss of the heme prosthetic group.


Subject(s)
FMN Reductase/antagonists & inhibitors , NADPH Oxidases/antagonists & inhibitors , Nitric Oxide/pharmacology , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Saccharomyces cerevisiae/enzymology , Enzyme Induction , FMN Reductase/biosynthesis , Models, Biological , Saccharomyces cerevisiae/drug effects
10.
J Biol Chem ; 277(42): 39450-5, 2002 Oct 18.
Article in English | MEDLINE | ID: mdl-12177066

ABSTRACT

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.


Subject(s)
Escherichia coli/enzymology , FMN Reductase/antagonists & inhibitors , Flavin-Adenine Dinucleotide/chemistry , Binding Sites , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Electrons , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , FMN Reductase/metabolism , Flavin-Adenine Dinucleotide/metabolism , Hydrogen , Kinetics , Mass Spectrometry , Models, Chemical , Protein Binding , Protein Conformation , Spectrometry, Fluorescence , Time Factors
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