Abstract
Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (km=68 microm) and trans-2-hexenoyl-CoA (km=91 microm). In the crotonyl-CoA-dependent reaction, both NADH (km=109 microm) or NADPH (km=119 microm) were accepted, with 2-3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.
Highlights
The mitochondrion of the photosynthetic flagellate Euglena gracilis is a facultatively anaerobic organelle that produces ATP under aerobic and anaerobic conditions
Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation
Purification and Microsequencing of Euglena TER—Early work on E. gracilis trans-enoyl-CoA reductase uncovered its involvement in mitochondrial fatty acid synthesis wax ester formation under anaerobic conditions [4, 15]
Summary
Crude extract 30% ammonium sulfate cut and dialysis DEAE Fractogel Phenyl-Sepharose Reactive red Hydroxyapatite Mono Q Native PAGE Superdex nmol. Enoyl-CoA reductase (EC 1.3.1.44), designated here as TER, reduces the double bond in enoyl-CoA to produce acyl-CoA [15]. In -oxidation this step is oxidative and irreversible under physiological conditions because acyl-CoA dehydrogenase (mitochondrial -oxidation) and acyl-CoA oxidase (peroxisomal -oxidation) are both linked to O2 reduction [20, 21]. We report the localization, purification, mass spectrometry sequencing, cloning, heterologous expression in E. coli, and kinetic parameters of TER from Euglena mitochondria
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