Abstract
Chain elongation of fatty acids is an important cellular process and is believed to occur in the endoplasmic reticulum of all eukaroytic cells. Herein we describe the cloning and characterization of a peroxisomal NADPH-specific trans-2-enoyl-CoA reductase, the key enzyme for a proposed peroxisomal chain elongation pathway. The reductase was solubilized and partially purified from guinea pig liver peroxisomes by affinity chromatography. On SDS-polyacrylamide gel electrophoresis, a 40-kDa band was identified as the enzyme, and its partial amino acid sequence (27 amino acids) was determined. A full-length cDNA for the reductase was cloned from a guinea pig liver cDNA library. The open reading frame of this nucleotide sequence encodes a 302-amino acid polypeptide with a calculated molecular mass of 32.5 kDa. Full-length mouse and human cDNA clones encoding homologous proteins have also been isolated. All of these translated polypeptides have the type I peroxisomal targeting signal, AKL, at the carboxyl terminus. The identity of the cloned enoyl-CoA reductase cDNAs was confirmed by expressing the guinea pig and human cDNAs in Escherichia coli. The His-tagged recombinant enzymes were found to have very high NADPH-specific 2-enoyl-CoA reductase activity with similar properties and specificity as the liver peroxisomal reductase. Both the natural and the recombinant enzyme catalyze the reduction of trans-2-enoyl-CoAs of varying chain lengths from 6:1 to 16:1, having maximum activity with 10:1 CoA. Northern blot analysis demonstrated that a single transcript of 1.3 kilobases is present in most mouse tissues, with particularly high concentrations in liver and kidney.
Highlights
In mammalian cells, the fatty acid chain elongation system has been shown to be present in both endoplasmic reticulum (ER)1 and mitochondria [1]
The mitochondrial chain elongation system is a reversal of the fatty acid -oxidation system; acetyl-CoA, instead of malonylCoA, is used for the condensation reaction, and the resulting -keto acyl-CoA undergoes the same series of reactions as described above [3]
The product of the enzymatic reaction was identified as a saturated fatty acyl-CoA. These two assay methods were qualitatively similar, lower activity (ϳ50%) was seen with the radiometric assay (Table I), indicating an isotope effect when 3H-labeled NADPH was used as the reductant
Summary
The incubation mixtures contained potassium phosphate buffer (50 mM), bovine serum albumin (2 mg/ml), either S-[4-3H]NADPH (70 M) or NADPH (0.1 mM), guinea pig liver peroxisomes (10 – 40 g of protein), and trans-2-enoyl CoA (30 M) as indicated below. Concentration of NADH, when used, was 0.1 mM. The incubations and assays were done as described. The average (n ϭ 3 or 4) specific activities along with their S.D. values are shown
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