ABSTRACT
Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.
Subject(s)
Carbohydrate Metabolism , Euglena gracilis/enzymology , Glucans/chemistry , UTP-Glucose-1-Phosphate Uridylyltransferase/chemistry , Catalysis , Glucans/metabolism , Kinetics , Protein Domains , UTP-Glucose-1-Phosphate Uridylyltransferase/metabolismABSTRACT
The removal, uptake and toxicity of chromium in Euglena gracilis cultured in absence and presence of malate with Cr(VI) or Cr(III) was evaluated. The malate extrusion and the extra- and intracellular Cr(VI) reduction capacity were determined and the contents of molecules with thiol group and ascorbate were also evaluated. Absence of malate in the medium decreased cell growth, increased Cr(III) toxicity, induced faster Cr(VI) disappearance from medium, and increased intracellular and intramitochondrial chromium accumulation. Both chromium species induced soluble and particulate ascorbate-dependent chromate reductase activities. Cells also secreted large amounts of malate and increased intracellular contents of thiol-molecules to bind extracellular and intracellular Cr(III), respectively. The former process was supported by significant increase in malate-producing enzyme activities and the assessment of the Cr-complexes indicated the in situ formation with thiol-molecules. The present results establish new paradigms regarding chromium stress on algae-like microorganisms: (i) Cr(III) may be more toxic than Cr(VI), depending on the culture (or environmental) conditions; (ii) several simultaneous mechanisms are turned on to inactivate chromium species and their toxic effects. These mechanisms, now well understood may further optimize, by genetically modifying E. gracilis, and facilitate the development of strategies for using this protist as potential bio-remediator of chromium-polluted water systems.
Subject(s)
Chromium/isolation & purification , Euglena gracilis/metabolism , Ascorbic Acid/metabolism , Chromium/metabolism , Culture Media , Enzyme Induction , Euglena gracilis/enzymology , Oxidoreductases/biosynthesisABSTRACT
To assess the toxic effect of Cr on energy metabolism, heterotrophic Euglena gracilis was grown in a medium that prompts high yield biomass and in the presence of different Cr(VI) or Cr(III) concentrations. The cell growth IC50 value was 12 and >250µM for Cr(VI) and Cr(III), respectively; in these cells chromium was accumulated and a fraction compartmentalized into mitochondria, and synthesis of cysteine and glutathione was induced. Respiration of control isolated mitochondria was strongly inhibited by added Cr(VI) or Cr(III) with L-lactate or succinate as substrates. In turn, cellular and mitochondrial respiration, respiratory Complexes I, III and IV, glycolysis and cytosolic NAD(+)-alcohol and -lactate dehydrogenases from cells cultured with Cr(VI) were significantly lower than control, whereas AOX and external NADH dehydrogenase activities were unaltered or increased, respectively. Addition of Cr(VI) or Cr(III) to isolated mitochondria or cytosol from control- or Cr(VI)-grown cells induced inhibition of respiration, respiratory Complexes III, IV and AOX, and glycolytic pyruvate kinase; whereas Complex I, external NADH dehydrogenase, and other glycolytic enzymes were unaffected. Protein contents of mitochondrial Complexes I, III, IV and V, and ANT were diminished in Cr(VI)-grown cells. Decreased respiration and glycolysis induced by Cr(VI) resulted in lower cellular ATP content. Results suggested that Cr(VI) cytotoxicity altered gene expression (as widely documented) and hence enzyme content, and induced oxidative stress, but it was also related with direct enzyme inhibition; Cr(III) was also cytotoxic although at higher concentrations. These findings establish new paradigms for chromium toxicity: Cr(VI) direct enzyme inhibition and non-innocuous external Cr(III) toxicity.
Subject(s)
Chromium/toxicity , Energy Metabolism/drug effects , Environmental Pollutants/toxicity , Euglena gracilis/drug effects , Animals , Carbon/metabolism , Cell Enlargement/drug effects , Chromium/metabolism , Euglena gracilis/enzymology , Euglena gracilis/metabolism , Glucans/metabolism , Glycolysis/drug effects , Heterotrophic Processes , Malondialdehyde/metabolism , Mitochondria/drug effects , Mitochondria/enzymologyABSTRACT
To identify some of the mechanisms involved in the high resistance to Cd(2+) in the protist Euglena gracilis, we studied the effect of Cd(2+) exposure on its energy and oxidative stress metabolism as well as on essential heavy metals homeostasis. In E. gracilis heterotrophic cells, as in other organisms, CdCl(2) (50 microM) induced diminution in cell growth, severe oxidative stress accompanied by increased antioxidant enzyme activity and strong perturbation of the heavy metal homeostasis. However, Cd(2+) exposure did not substantially modify the cellular respiratory rate or ATP intracellular level, although the activities of respiratory complexes III and IV were strongly decreased. In contrast, an enhanced capacity of the alternative oxidase (AOX) in both intact cells and isolated mitochondria was determined under Cd(2+) stress; in fact, AOX activity accounted for 69-91% of total respiration. Western blotting also revealed an increased AOX content in mitochondria from Cd(2+)-exposed cells. Moreover, AOX was more resistant to Cd(2+) inhibition than cytochrome c oxidase in mitochondria from control and Cd(2+)-exposed cells. Therefore, an enhanced AOX seems to be a relevant component of the resistance mechanism developed by E. gracilis against Cd(2+)-stress, in addition to the usual increased antioxidant enzyme activity, that enabled cells to maintain a relatively unaltered the energy status.
Subject(s)
Algal Proteins/metabolism , Antioxidants/metabolism , Cadmium/toxicity , Electron Transport Complex III/metabolism , Electron Transport Complex I/metabolism , Euglena gracilis/enzymology , Oxidoreductases/metabolism , Protozoan Proteins/metabolism , Stress, Physiological/drug effects , Animals , Homeostasis/drug effects , Mitochondrial Proteins , Oxygen Consumption/drug effects , Plant ProteinsABSTRACT
Some kinetic and thermodynamic properties of the plasma membrane adenylyl cyclase (AC) from the protist Euglena gracilis were examined. The AC kinetics for Mg-ATP was hyperbolic with a K(m) value of 0.33-0.43 mM, whereas the inhibition exerted by 2('),5(')-dideoxyadenosine was of the mixed type with a K(i) of 80-147 microM. The V(m) value (0.9 or 1.8 nmol(mg protein)(-1)min(-1)) changed, depending upon the carbon source in the growth medium (lactic acid or glutamate plus malate). Lactic acid membrane AC was slightly more thermolabile (from 28 to 40 degrees C) and showed higher activation energy (range 15-25 degrees C). With lactate, the total and saturated fatty acid percentage content in the plasma membrane was significantly greater than with glutamate plus malate, whereas the percentage content of polyunsaturated (n-3) fatty acids was lower. The data suggest that the fatty acid composition, as changed by the carbon source in the growth medium, may modulate the AC activity in Euglena.
Subject(s)
Adenylyl Cyclases/chemistry , Adenylyl Cyclases/metabolism , Euglena gracilis/enzymology , Adenosine Triphosphate/metabolism , Animals , Cell Membrane/chemistry , Cell Membrane/enzymology , Cyclic AMP/metabolism , Enzyme Stability , Fatty Acids/analysis , Kinetics , Signal Transduction , ThermodynamicsABSTRACT
The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for L- and D-lactate were similar, but the V(max) was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both L- and D-lactate. Oxalate exerted a mixed-type inhibition regarding L- or D-lactate and also against DCPIP. The rate of L-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of L-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability at 70 degrees C and denaturation of L-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.
Subject(s)
Euglena gracilis/enzymology , L-Lactate Dehydrogenase/metabolism , Lactate Dehydrogenases , Lactic Acid/metabolism , Mitochondria/enzymology , Animals , Binding, Competitive , Biological Transport , Enzyme Stability , Kinetics , L-Lactate Dehydrogenase (Cytochrome) , Membrane Proteins/metabolism , Molecular Weight , Solubility , StereoisomerismABSTRACT
The kinetic properties of the enzyme L-glutamate:4,5-dioxovaleric acid aminotransferase (Glu:DOVA transaminase) from Euglena gracilis have been studied. 5-Aminolevulinic acid formation was linear with time for at least 45 min at 37 degrees C and L-glutamate was the most effective amino-group donor. Lineweaver-Burk double-reciprocal plots suggested a ping-pong reaction mechanism, with Km values for L-glutamate and DOVA of 1.92 mM and 0.48 mM respectively. Competitive parabolic substrate inhibition by DOVA at concentrations greater than 3.5-4.5 mM was observed. Glyoxylate (4-10 mM) was found to be a competitive inhibitor with respect to DOVA, whereas at low concentrations (0-4 mM) noncompetitive plots were obtained. An analysis of the possible enzyme forms involved, was carried out. In more crude preparations most of the enzyme is found to be in the form of an enzyme-glutamate complex.