Peroxisomal Delta3-cis-Delta2-trans-enoyl-CoA isomerase encoded by ECI1 is required for growth of the yeast Saccharomyces cerevisiae on unsaturated fatty acids.
We have identified the Saccharomyces cerevisiae gene ECI1 encoding Delta3-cis-Delta2-trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the beta-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 Delta3-cis-Delta2-trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.
Gurvitz
A
A
Institut für Biochemie und Molekulare Zellbiologie der Universität Wien and Ludwig Boltzmann Forschungsstelle für Biochemie, Vienna Biocenter, Dr. Bohrgasse 9, A-1030 Wien, Austria.
Mursula
A M
AM
Firzinger
A
A
Hamilton
B
B
Kilpeläinen
S H
SH
Hartig
A
A
Ruis
H
H
Hiltunen
J K
JK
Rottensteiner
H
H
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002451:Cell Compartmentation; D017124:Conserved Sequence; D004746:Enoyl-CoA Hydratase / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004790:Enzyme Induction; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins; D007535:Isomerases / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007536:Isomerism; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D008830:Microbodies / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000172:deficiency / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D019301:Oleic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D063994:Peroxisomal Bifunctional Enzyme; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D020218:Response Elements; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D017386:Sequence Homology, Amino Acid
D005231:Fatty Acids, Unsaturated; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019301:Oleic Acid; D019308:Palmitic Acid; D015094:3-Hydroxyacyl CoA Dehydrogenases; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases