Molecular Characterization of Saccharomyces cerevisiae Δ3,Δ2-Enoyl-CoA Isomerase

Abstract

We report here the identification of the Saccharomyces cerevisiae peroxisomal ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase, an enzyme that is essential for the ␤-oxidation of unsaturated fatty acids. The yeast gene YLR284C was identified in an in silico screen for genes that contain an oleate response element, a transcription factor-binding site common to most fatty acid-induced genes. Growth on oleic acid resulted in a significant increase in YLR284C mRNA, demonstrating that it is indeed an oleate-induced gene. The deduced product of YLR284C contains a type 1 peroxisomal targeting signal-like sequence at its C terminus and localizes to the peroxisome in a PEX8-dependent manner. Removal of YLR284C from the S. cerevisiae genome eliminated growth on oleic acid, but had no effect on peroxisome biogenesis, indicating a role for YLR284C in fatty acid metabolism. Cells lacking YLR284C had no detectable ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase activity, and a bacterially expressed form of this protein catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 16 units/mg. We conclude that YLR284C encodes the yeast peroxisomal ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase and propose a new name, ECI1, to reflect its enoyl-CoA isomerase activity.

Identification and Characterization of HAOX1, HAOX2, and HAOX3, Three Human Peroxisomal 2-Hydroxy Acid Oxidases

“…Both sets of primers append BamHI and NotI sites (underlined) to the 5Ј-and 3Ј-ends of the HAOX1 ORF, respectively. The PCR product from each reaction was digested with BamHI and NotI and cloned between the BamHI and NotI sites of pMBP (27), a modified form of the pMALc2 expression vector (New England Biolabs) to make pMBP-HAOX1 and pMBP-HAOX1⌬KI, respectively. The HAOX1 ORF was excised from pMBP-HAOX1 and inserted between the BamHI and NotI sites of pcDNA3-Nmyc (28), a mammalian expression vector designed to express proteins in fusion with an amino-terminal c-myc epitope, creating pcDNA3-Nmyc/HAOX1.…”

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

“…All polymerase chain reactions were performed with a low error-rate mixture of polymerases (Expand, Roche Molecular Biochemicals). The polymerase chain reaction product from each reaction was digested with SalI and NotI and subcloned into the SalI and NotI sites of pMBP (7,16). The sequence of each form of the human PECI ORF in pMBP was confirmed by automated fluorescent sequencing, and the resulting plasmids were denoted pMBP-PECI and pMBP-PECI⌬SKL.…”

“…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.…”

Peroxisomes: simple in function but complex in maintenance

“…Cytosolic and peroxisomal NADP-dependent isocitrate dehydrogenases might function in a shuttle in a manner similar to the malate dehydrogenases. The goal is different, however -to keep the intraperoxisomal pool of NADP reduced [12][13][14] . This results in the simultaneous flow of reduction equivalents in opposite directions, which are kept separated by different cofactors (NAD/NADP) and substrates (malate/citrate).…”