Molecular cloning and expression of mammalian peroxisomal trans-2-enoyl-coenzyme A reductase cDNAs

Chain elongation of fatty acids is an important cellular process and is bel ieved to occur in the endoplasmic reticulum of all eukaroytic cells. Herein we describe the cloning and characterization of a peroxisomal NADPH-specif ic trans-2-enoyl-CoA reductase, the key enzyme for a proposed peroxisomal c hain elongation pathway. The reductase was solubilized and partially purifi ed from guinea pig liver peroxisomes by affinity chromatography. On SDS-pol yacrylamide gel electrophoresis, a 40-kDa band was identified as the enzyme , and its partial amino acid sequence (27 amino acids) was determined. A fu ll-length cDNA for the reductase was cloned from a guinea pig liver cDNA li brary. The open reading frame of this nucleotide sequence encodes a 302-ami no acid polypeptide with a calculated molecular mass of 32.5 kDa. Full-leng th 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 h uman cDNAs in Escherichia coli. The His-tagged recombinant enzymes were fou nd to have very high NADPH-specific 2-enoyl-CoA reductase activity with sim ilar properties and specificity as the liver peroxisomal reductase. Both th e natural and the recombinant enzyme catalyze the reduction of trans-2-enoy l-CoAs of varying chain lengths from 6:1 to 16:1, having maximum activity w ith 10:1 CoA. Northern blot analysis demonstrated that a single transcript of 1.3 kilobases is present in most mouse tissues, with particularly high c oncentrations in liver and kidney.