Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of beta-oxidation enzyme activities as well as a set of auxiliary activities to provide the beta-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of beta-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p-Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in beta-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in beta-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

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

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Fatty acids with double bonds at odd-numbered positions such as oleic acid can enter ␤-oxidation via a pathway relying solely on the auxiliary enzyme ⌬ 3 -⌬ 2 -enoyl-CoA isomerase, termed the isomerase-dependent pathway. Two novel alternative pathways have recently been postulated to exist in mammals, and these additionally depend on ⌬ 3,5 -⌬ 2,4 -dienoyl-CoA isomerase (diisomerase-dependent) or on ⌬ 3,5 -⌬ 2,4 -dienoyl-CoA isomerase and 2,4-dienoyl-CoA reductase (reductase-dependent). We report the identification of the Saccharomyces cerevisiae oleic acid-inducible DCI1 (YOR180c) gene encoding peroxisomal di-isomerase. Enzyme assays conducted on soluble extracts derived from yeast cells overproducing Dci1p using 3,5,8,11,14-eicosapentenoyl-CoA as substrate demonstrated a specific diisomerase activity of 6 nmol ؋ min ؊1 per mg of protein. Similarly enriched extracts from eci1⌬ cells lacking peroxisomal 3,2-isomerase additionally contained an intrinsic 3,2-isomerase activity that could generate 3,5,8,11,14-eicosapentenoyl-CoA from 2,5,8,11,14-eicosapentenoyl-CoA but not metabolize trans-3-hexenoyl-CoA. Amplification of this intrinsic activity replaced Eci1p since it restored growth of the eci1⌬ strain on petroselinic acid for which di-isomerase is not required whereas Eci1p is. Heterologous expression in yeast of rat di-isomerase resulted in a peroxisomal protein that was enzymatically active but did not re-establish growth of the eci1⌬ mutant on oleic acid. A strain devoid of Dci1p grew on oleic acid to wild-type levels, whereas one lacking both Eci1p and Dci1p grew as poorly as the eci1⌬ mutant. Hence, we reasoned that yeast di-isomerase does not additionally represent a physiological 3,2-isomerase and that Dci1p and the postulated alternative pathways in which it is entrained are dispensable for degrading oleic acid.