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Novel mitochondrial alcohol metabolizing enzymes of Euglena gracilis

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Abstract

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.

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Acknowledgments

This work was supported in part by CONACyT grants No. 78775, 80534, 89412, 102926 and ICyTDF grant PICS08-5.

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  1. Departamento de Bioquímica, Instituto Nacional de Cardiología, Juan Badiano No. 1, Seccion XVI, Tlalpan, México DF, 14080, México

    Belem Yoval-Sánchez, Ricardo Jasso-Chávez, Elizabeth Lira-Silva, Rafael Moreno-Sánchez & José S. Rodríguez-Zavala

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  1. Belem Yoval-Sánchez
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  2. Ricardo Jasso-Chávez
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Correspondence to José S. Rodríguez-Zavala.

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Figure S1

Effect of acetaldehyde on the generation of Δψ driven by different substrates in isolated Euglena mitochondria. Transmembrane electrical potential was assayed as described under Materials and Methods. A, Collapse of malate-generated ∆ψ by acetaldehyde. Mal, 2 mM malate; Acetal, 0.1 mM acetaldehyde; Rote, 2 μM rotenone; D-Lac, 0.2 mM D-lactate; CCCP, 2 μM carbonylcyanide-3-chlorophenylhydrazone. B, Collapse of ethanol-generated ∆ψ by acetaldehyde and further Δψ generation by D-lactate. EtOH, 0.44 mM ethanol; Acetal, 0.1 mM acetaldehyde; Rote, 2 μM rotenone; D-Lac, 0.2 mM D-lactate; CCCP, 2 μM carbonylcyanide-3-chlorophenylhydrazone. C and D, Lack of effect of acetaldehyde on the ∆ψ generated by succinate semialdehyde (C) or D-Lactate (D). SSA, 0.25 mM succinate semialdehyde; Acetal, 0.1 mM acetaldehyde; D-Lac, 0.2 mM D-lactate; CCCP, 2 μM carbonylcyanide-3-chlorophenylhydrazone. Results are representative of at least 3 independent mitochondrial preparations. (JPEG 767 kb)

Figure S2

Kinetics of Euglena mmADH and mmALDH. Kinetic assays were carried out with an enriched mitochondrial membrane fraction prepared as indicated under Materials and Methods. A, Kinetic profile of mmADH showing two well defined components. B, Kinetic profile of mmALDH showing one hyperbolic component. In both graphs, the insets show the Lineweaver-Burk plot of the curves. Results are representative of experiments with at least 3 independent membrane preparations. (JPEG 464 kb)

Figure S3

Generation of ΔpH by SMPs. The generation of ΔpH by SMPs was assayed as detailed under Materials and Methods. A, ΔpH generation was assessed using 5 mM succinate as the substrate in the presence of rotenone. B, ΔpH generation in the absence of substrate added. C, generation of ΔpH by SMPs after the addition of 0.2 mM acetaldehyde in the presence of 0.1 mM pyrazole. Results are representative of experiments with at least 3 independent SMPs preparations. (JPEG 377 kb)

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Yoval-Sánchez, B., Jasso-Chávez, R., Lira-Silva, E. et al. Novel mitochondrial alcohol metabolizing enzymes of Euglena gracilis . J Bioenerg Biomembr 43, 519–530 (2011). https://doi.org/10.1007/s10863-011-9373-4

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