Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids*
- Aner Gurvitzद,
- Anu M. Mursula§,
- Andreas Firzinger‡,
- Barbara Hamilton‡,
- Seppo H. Kilpeläinen§,
- Andreas Hartig‡,
- Helmut Ruis‡,
- J. Kalervo Hiltunen§‖ and
- Hanspeter Rottensteiner‡
- From the ‡Institut für Biochemie und Molekulare Zellbiologie der Universität Wien and Ludwig Boltzmann Forschungsstelle für Biochemie, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Wien, Austria and the §Department of Biochemistry, Biocenter Oulu, University of Oulu, FIN-90570 Oulu, Finland
Abstract
We have identified the Saccharomyces cerevisiae gene ECI1 encoding Δ3-cis-Δ2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the β-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 ECI1transcript 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 totrans-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 Δ3-cis-Δ2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.
Footnotes
-
↵* This work was supported in part by Fonds zur Förderung der wissenschaftlichen Forschung (FWF) (Vienna, Austria) Grants P10604 and P12061 (to B. H.), Jubiläumsfonds der Österreichischen Nationalbank, Austria, Grant 6517 (to H. R.), and grants from the Sigrid Juselius Foundation, Finland, and the Academy of Finland (to J. K. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵¶ Supported by FWF Grant P12118.
-
↵‖ To whom correspondence should be addressed: Dept. of Biochemistry, University of Oulu, Linnanmaa, FIN-90570, Finland. Tel.: 358-8-553-1150; Fax: 358-8-553-1141; E-mail:kalervo.hiltunen{at}oulu.fi.
-
↵2 I. V. Karpichev, J. Lopez, and G. M. Small, manuscript in preparation.
- Abbreviations:
- MFE
-
multifunctional enzyme
- EMSA
-
electrophoretic mobility shift assay
- GFP
-
green fluorescent protein
- ORE
-
oleate response element
- PCR
-
polymerase chain reaction
- kb
-
kilobases.
-
- Received July 31, 1998.
- Revision received August 28, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
