PMID:15569691

Citation	Hoffmeister, M, Piotrowski, M, Nowitzki, U and Martin, W (2005) Mitochondrial trans-2-enoyl-CoA reductase of wax ester fermentation from Euglena gracilis defines a new family of enzymes involved in lipid synthesis. J. Biol. Chem. 280:4329-38
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.
Links	PubMed Online version:10.1074/jbc.M411010200
Keywords	Adenosine Triphosphate/chemistry; Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; Blotting, Western; Catalysis; Chromatography; Cloning, Molecular; Culture Media; DNA, Complementary/metabolism; Electrons; Electrophoresis, Polyacrylamide Gel; Escherichia coli/metabolism; Euglena gracilis/metabolism; Evolution, Molecular; Fatty Acids/metabolism; Kinetics; Lipid Metabolism; Mass Spectrometry; Mitochondria/enzymology; Molecular Sequence Data; NAD/metabolism; NADH, NADPH Oxidoreductases/metabolism; NADH, NADPH Oxidoreductases/physiology; NADP/metabolism; Nucleic Acid Hybridization; Open Reading Frames; Operon; Oxidoreductases Acting on CH-CH Group Donors; Peptides/chemistry; Phylogeny; Protein Binding; Protein Structure, Tertiary; Silver Staining; Trypsin/chemistry
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