Abstract
rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts that rECH1p has the hydratase fold in the core domain and two domains for interaction with other subunits. When we incubated 3,5,8,11, 14-eicosapentaenoyl-CoA with purified rECH1p, the spectral data suggested a switching of the double bonds from the Delta3-Delta5 to the Delta2-Delta4 positions. This was confirmed by demonstrating that the product was a valid substrate for 2,4-dienoyl-CoA reductase. These results indicate that rECH1p is Delta3,5-Delta2,4-dienoyl-CoA isomerase. Subcellular fractionation and immunoelectron microscopy using antibodies to a synthetic polypeptide derived from the C terminus of rECH1p showed that rECH1p is located in the matrix of both mitochondria and peroxisomes in rat liver. Consistent with these observations, the 36,000-Da rECH1p has a potential N-terminal mitochondrial targeting signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the targeting signal results in a mature mitochondrial form with a molecular mass of 32,000 Da; transport to peroxisomes yields a protein of 36,000 Da.
Highlights
We show that the rECH1 protein is a ⌬3,5-⌬2,4-dienoylCoA isomerase participating in the metabolism of unsaturated fatty acids with double bonds at the ⌬5 position
In control incubations with arachidoyl-CoA, these changes were not observed (Fig. 4, C and D), indicating that the substrate for 2,4-dienoyl-CoA reductase was produced only in the initial incubation with arachidonoyl-CoA. These results show that rECH1 protein (rECH1p) transfers the conjugated double bonds from ⌬3,5 to ⌬2,4 positions of enoyl-CoAs, and rECH1p is a ⌬3,5-⌬2,4dienoyl-CoA isomerase
Published studies on the distribution of the ⌬3,5-⌬2,4-dienoylCoA isomerase in the rat including subcellular fractionation, immunoblotting, immunoelectron microscopy, enzyme activity measurements, and protein purification provide evidence that the dienoyl-CoA isomerase activity in liver is associated with both peroxisomes and mitochondria [11,12,13,14, 44]
Summary
(Received for publication, August 18, 1997, and in revised form, October 6, 1997). S. Ate in both peroxisomal and mitochondrial -oxidation and since the double bonds of natural unsaturated fatty acids are mostly in the cis configuration at either odd- or even-numbered positions, auxiliary enzymes are required to link them to the -oxidation pathway These auxiliary activities include 2,4enoyl-CoA reductase (EC 1.3.1.34), ⌬3-⌬2-enoyl-CoA isomerase (EC 5.3.3.8), and ⌬3,5-⌬2,4-dienoyl-CoA isomerase. We show that the rECH1 protein (rECH1p) is a ⌬3,5-⌬2,4-dienoylCoA isomerase participating in the metabolism of unsaturated fatty acids with double bonds at the ⌬5 position. This is the first characterization of a dienoyl-CoA isomerase at the molecular level
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