| Δ3-Δ2-Enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae: Molecular and structural characterization | ||
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Northern analysis revealed that ECI1 transcripts accumulate when the cells are grown on oleate, whereas no detectable gene expression could be seen when glucose was used as the carbon source. Furthermore, according to EMSA studies, the regulation is mediated by the binding of the transcription factors Pip2p and Oaf1p (Luo et al. 1996, Rottensteiner et al. 1996) to the ORE in the promoter region of the gene.
To study the potential role of Eci1p in fatty acid degradation, an ECI1-deleted yeast strain was grown on saturated and unsaturated fatty acids as the sole carbon source. The ECI1 knock-out was able to utilize the saturated fatty acid, palmitic acid, suggesting that Eci1p does not participate in the β -oxidation of straight-chain fatty acids. Instead, the ECI1-disrupted cells did not grow on any of the three unsaturated fatty acids tested. This indicated that Eci1p is involved in the metabolism of double bonds in both odd- and even-numbered positions of fatty acids, possibly acting as a Δ3-Δ2-enoyl-CoA isomerase. The function of Eci1p was tested further by supplying the mutant yeast cells with the rat peroxisomal Δ3-Δ2-enoyl-CoA isomerase, which is part of MFE-1, also containing 2–enoyl-CoA hydratase-1 and L-specific 3-hydroxyacyl-CoA dehydrogenase activities (Palosaari & Hiltunen 1990). The Δ3-Δ2-enoyl-CoA isomerase activity of MFE-1 could restore the growth of ECI1-deleted cells on oleic acid, suggesting that ECI1 encodes for an Δ3-Δ2-enoyl-CoA isomerase.
For further characterization, the full-length Eci1p, consisting of 280 amino acid residues, was expressed as a recombinant protein in E. coli and purified. Analysis on SDS-PAGE showed one single protein band corresponding to a molecular mass of 32 kDa, which is in agreement with the molecular mass calculated on the basis of the amino acid sequence of Eci1p, 31.7 kDa. Eci1p eluted from the gel filtration column in the same elution volume as the rat Δ3,5-Δ2,4-dienoyl-CoA isomerase, which has a native mass of 170 kDa (Filppula et al. 1998). In addition, according to dynamic light scattering analysis, the approximate molecular mass is 151 kDa. From these results, it could be concluded that, in solution, Eci1p is a hexameric protein formed of six identical 32-kDa subunits. The purified Eci1p was assayed for Δ3-Δ2-enoyl-CoA isomerase, Δ3,5-Δ2,4-dienoyl-CoA isomerase and 2-enoyl-CoA hydratase-1 activities. No dienoyl-CoA isomerase or hydratase-1 activity could be detected, whereas the purified protein was found to possess a specific Δ3-Δ2-enoyl-CoA isomerase activity of 11.2 µmol/min/mg (kcat 6.0 sec-1) and a Km value of 21.5 µM for trans-3-hexenoyl-CoA. Eci1p is thus a monofunctional enoyl-CoA isomerase and will be referred to as the yeast Δ3-Δ2-enoyl-CoA isomerase.
The amino acid sequence of the yeast enoyl-CoA isomerase was aligned with the sequences of some other hydratase/isomerase superfamily members, hydratase-1 (Minami-Ishii et al. 1989), mitochondrial enoyl-CoA isomerase (Müller-Newen & Stoffel 1991, Palosaari et al. 1991), 4-CBA-CoA dehalogenase (Babbitt et al. 1992) and Δ3,5-Δ2,4-dienoyl-CoA isomerase (FitzPatrick et al. 1995, Filppula et al. 1998), in order to make suggestions for the possible catalytic residues of the yeast enoyl-CoA isomerase. Surprisingly, the active site amino acid of hydratase-1, Glu164 (Müller-Newen et al. 1995), which is also present in the mitochondrial enoyl-CoA isomerase, was not conserved in the yeast enoyl-CoA isomerase, but was replaced by a phenylalanine. Instead, we suggested that a protic residue close to that, Tyr148, could be involved in the proton exchange. Another possibility was determined to be Glu158, which corresponds to the catalytic residues of the dienoyl-CoA isomerase and the 4-CBA-CoA dehalogenase, Asp204 (Modis et al. 1998) and Asp145 (Benning et al. 1996, Yang et al. 1996), respectively. Both Tyr148 and Glu158 were separately exchanged to alanine, and the enoyl-CoA isomerase activity of the mutated enzymes was measured. The enoyl-CoA isomerase activity of Tyr148Ala was comparable to the activity of the wild type (unpublished results), whereas the Glu158Ala variant lacked isomerase activity completely, suggesting that Glu158 is involved in the reaction mechanism of the yeast enoyl-CoA isomerase.
For the determination of its subcellular localization, Eci1p was tagged with GFP, and the location in both wild-type cells and pex6Δ cells lacking peroxisomes was examined by fluorescence microscopy. In the wild-type cells, GFP-Eci1p was detected as punctate fluorescence indicating compartmentalization. In the pex6Δ cells, instead, the fluorescence was diffuse due to the location of GFP-Eci1p in the cytosol. DNA staining, which showed intact mitochondria in both yeast cell types, excluded the localization of the fusion protein in the mitochondria. This led to the conclusion that Eci1p is located in yeast peroxisomes.