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The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd(2+)) and biochemically characterized. High biomass (8.5×10(6)cellsmL(-1)) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O₂, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25-33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd(2+) which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd(2+) induced a higher MDA production. Cd(2+) stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd(2+) from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd(2+) under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O₂ concentration is particularly low., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64-6.5 mM for ethanol, and 0.16-0.88 mM for NAD+, while the ALDH Km values were 1.7-5.3 μM for acetaldehyde and 33-47 μM for NAD+. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Photosynthetic Euglena gracilis grown with different K(2)CrO(4) concentrations was analyzed for its ability to take up, retain and reduce Cr(VI). For comparison, cells were also exposed to CrCl(3). Cellular Cr(VI) uptake at pH 7.2 showed a hyperbolic saturation pattern with K (m) of 1.1 mM, V (m) of 16 nmol (h x 10(7) cells)(-1), and K (i sulfate) of 0.4 mM. Kinetic parameters for sulfate uptake were similar, K (m) = 0.83 mM, V (m) = 15.9 nmol (h x 10(7)cells)(-1) and K (i chromate) = 0.3 mM. The capacity to accumulate chromium depended on the ionic species, external concentration and pH of the incubation medium. Cr(VI) or Cr(III) accumulation was negligible in the acidic (pH 3.5) culture medium, in which Cr(VI) was abiotically reduced to Cr(III). At pH 7.2 Cr(VI) was fully stable and high accumulation (>170 nmol/1 x 10(7) cells at 1 mM K(2)CrO(4)) was achieved; surprisingly, Cr(III) accumulation was also significant (>35 nmol/1 x 10(7) cells at 1 mM CrCl(3)). Cr(VI) was reduced by cells at pH 7.2, suggesting the presence of an external reductive activity. Cr(VI) induced an increased cysteine and glutathione content, but not in phytochelatins suggesting that chromium accumulation was mediated by monothiol compounds.

Euglena gracilis is a freshwater free-living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over-expression, and biochemical characterization of a medium-chain NAD(+)-dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three-dimensional model, all the residues involved in Zn(2+), cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over-expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V(m) of 11.7+/-3.6 U/mg protein and a K(m) of 3.2+/-0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD(+) with a K(m) value of 0.39+/-0.1 mM and Mg(2+) or Zn(2+) were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short- and long-chain alcohols in this microorganism.

Rhodoquinone (RQ) participates in fumarate reduction under anaerobiosis in some bacteria and some primitive eukaryotes. Euglena gracilis, a facultative anaerobic protist, also possesses significant rhodoquinone-9 (RQ9) content. Growth under low oxygen concentration induced a decrease in cytochromes and ubiquinone-9 (UQ9) content, while RQ9 and fumarate reductase (FR) activity increased. However, in cells cultured under aerobic conditions, a relatively high RQ9 content was also attained together with significant FR activity. In addition, RQ9 purified from E. gracilis mitochondria was able to trigger the activities of cytochrome bc1 complex, bc1-like alternative component and alternative oxidase, although with lower efficiency (higher Km, lower Vm) than UQ9. Moreover, purified E. gracilis mitochondrial NAD+-independent D-lactate dehydrogenase (D-iLDH) showed preference for RQ9 as electron acceptor, whereas L-iLDH and succinate dehydrogenase preferred UQ9. These results indicated a physiological role for RQ9 under aerobiosis and microaerophilia in E. gracilis mitochondria, in which RQ9 mediates electron transfer between D-iLDH and other respiratory chain components, including FR.

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD(+)-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K(+) or NH(4)(+). Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.

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.

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., (Copyright 2001 Academic Press.)