Mitochondrial 3-2trans-Enoyl-CoA Isomerase. Purification, Cloning, Expression, and Mitochondrial Import of the Key Enzyme of Unsaturated Fatty acidβ-Oxidation

Abstract

3-2iriws-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial ß-oxidation of unsaturated fatty acids in bacteria, plant and animal cells. The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two Chromatographie steps. Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation. Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries. 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32254 Da.The 1.3-kb mRNAis most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain. In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies. In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2fra«s-enoyl-CoA isomerase with 264 amino-acid residues (M r 29706). A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of M r 30475.

Oxidoreductase, morphogenesis, extracellular matrix, and calcium ion-binding gene expression in streptozotocin-induced diabetic rat heart

“…Among genes with more modest degrees of increased expression (1.5-to 2-fold change), three play especially important roles in cellular energetics. Dci catalyzes the interconversion of 3-cis-dodecenoyl-CoA and 2-trans-dodecenoyl-CoA, a key step in fatty acid ␤-oxidation (42). Hadha and Hadhb are the ␣-and ␤-subunits of the liver mitochondrial fatty acid oxidation multienzyme complex (29).…”

Disruption of Mitochondrial β-Oxidation of Unsaturated Fatty Acids in the 3,2-trans-Enoyl-CoA Isomerase-deficient Mouse

“…Dodec-3-and 9-cis-ene-1,6-dioic acid is then activated by the microsomal dicarboxylyl-CoA synthase (32) and further degraded either in mitochondria or in peroxisomes to hex-3-cis-ene-1,6-dicarboxylic acid. Octadi-2,5-cis-ene-1,8-dioic acid is another characteristic and abundant degradation product, which is derived from linoleic acid (18: 2 9,12 ). Degradation of octadi-2,5-cis-ene-1,8-dioic acids via peroxisomal ␤-oxidation complex (hydratase to the D-3-hydroxyand epimerase to the L-3-hydroxy-intermediate, dehydrogenase, and thiolase) might further increase the hex-3-cis-ene-1,6-dioic acid concentration in the urinary dicarboxylic acid pattern.…”

“…of oleic acid (18:1 9 ), linoleic acid (18:1 9,12 ), and ␣-linolenic acid (18:1 9,12,15 ), yields 3-cis-enoyl-CoA-intermediates. They are isomerized by the mitochondrial 3,2-transenoyl-CoA isomerase (ECI) 1 (EC 5.3.3.8) to their respective 2-trans-enoyl-CoA isomers, common substrates of enoyl-CoA hydratase of the ␤-oxidation cycle of saturated fatty acyl-CoA esters (1).…”

Molecular cloning and gene organization of the mouse mitochondrial 3,2‐trans‐enoyl‐CoA isomerase

“…Isolation ofmECI cDNA and genomic clones An oligo-dT primed mouse liver cDNA library in jlgtl l(Clontech) was screened with rat mECI cDNA described previously [6]. Plaques producing positive signals were isolated and the phage DNA prepared by established procedures [12].…”

“…isomeruse cDNA A 900 bp EcoRI fragment of the previously cloned rat liver mEC1 [6] was used for screening a mouse liver cDNA library in agt 11. A cDNA clone with a 1 kb insert spanning the whole coding region of mEC1 were isolated and the sequence determined.…”