Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

Abstract

We have identified the Saccharomyces cerevisiae gene ECI1 encoding ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the ␤-oxidation of (poly)-unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32,000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.In mammals, degradation of fatty acids via ␤-oxidation occurs in both peroxisomes and mitochondria; however, in yeast this is solely a peroxisomal process (1). The yeast Saccharomyces cerevisiae is capable of degrading saturated fatty acids in a multistep process catalyzed by the enzymes acyl-CoA oxidase (Pox1p), 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacylCoA dehydrogenase (Fox2p), and 3-ketoacyl-CoA thiolase (Pot1p/Fox3p) as shown in Fig. 1 (Refs. 2-5).The only double bonds of unsaturated intermediates that are catabolized via the classical ␤-oxidation pathway are at the ⌬ 2 -position and in the trans-configuration. Therefore, the removal of other double bonds requires the participation of auxiliary enzymes. Fatty acids with double bonds at odd-numbered positions such as oleic acid (cis-C 18:1(9) ) require for their degradation (along with the regular ␤-oxidation enzymes) ⌬ 3

Characterization of PECI, a Novel Monofunctional Δ3,Δ2-Enoyl-CoA Isomerase of Mammalian Peroxisomes

“…We observed that recombinant PECI catalyzed the isomerization 3-cis-octenoyl-CoA to 2-trans-enoyl-CoA with a specific activity of 27 units/mg of protein. (7,8). We also assayed recombinant PECI for 2-enoyl-CoA hydratase and ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase activities (Table I) but were unable to detect these activities using crotonyl-CoA and 3,5-octadienoyl-CoA as the respective substrates.…”

“…However, we and others have recently identified and characterized the S. cerevisiae ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase (7,8) and have found that this enzyme, Eci1p, shares few primary sequence features with MFE1. In this report we describe the identification and characterization of a novel, ubiquitously expressed mammalian peroxisomal ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase (PECI) that is homologous to yeast Eci1p.…”

“…Although these enzymes are necessary for all fatty acid ␤-oxidation, the complete metabolism of unsaturated fatty acids requires one or more additional auxiliary enzymes (6). These include ⌬ 3 ,⌬ 2 -enoylCoA isomerase (Eci1p) (7,8), a 2,4-dienoyl-CoA reductase (Sps19p) (9), an NADP ϩ -dependent isocitrate dehydrogenase (Idp3p) (10,11), and a ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase (although this enzyme has yet to be identified in yeast). Among these auxiliary enzymes, ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase (Eci1p) is unique in that its activity is essential for the ␤-oxidation of all unsaturated fatty acids ( Fig.…”

Alternatives to the Isomerase-dependent Pathway for the β-Oxidation of Oleic Acid Are Dispensable in Saccharomyces cerevisiae

“…1A (2)(3)(4)(5). To metabolize evennumbered cis-double bonds in unsaturated fatty acids such as petroselinic acid (cis-C 18:1 (6) ), yeast rely on peroxisomal Sps19p (6) which corresponds to the auxiliary enzyme 2,4-dienoyl-CoA reductase (2,4-reductase) and peroxisomal Eci1p (7,8) representing the auxiliary enzyme ⌬ 3 -⌬ 2 -enoyl-CoA isomerase (3,2-isomerase; Fig. 1B).…”

iTRAQ-based quantitative proteomic analysis of longissimus muscle from growing pigs with dietary supplementation of non-starch polysaccharide enzymes

“…Protein species related to lipid metabolism were also up-regulated in pigs fed NSPEs. Of these proteins, mitochondrial δ3,δ2-dienoyl-CoA isomerase (ECI1) acts as an auxiliary enzyme in the β-oxidation of polyunsaturated fatty acids (Gurvitz et al, 1998). Histidine triad nucleotide binding protein 2 (HINT2) is positively responsible for regulating mitochondrial lipid metabolism and respiration in the livers of mice (Martin et al, 2013).…”