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Papers In Press, published online ahead of print May 12, 2000
Neuroscience Laboratory, University of Michigan, Ann Arbor, MI 48104-1687
Corresponding Author: akhajra{at}umich.edu
Chain elongation of fatty acids is an important cellular process and is believed to occur in the endoplasmic reticulum of all eukaroytic cells. Although a peroxisomal chain elongation pathway has been described, definitive evidence had been lacking. Herein we describe the cloning and characterization of a peroxisomal NADPH-specific trans-2-enoyl CoA reductase, the key enzyme for the proposed chain elongation pathway. The reductase was solubilized and partially purified from guinea pig liver peroxisomes by affinity chromatography. On SDS-PAGE a 40 kDa band was identified as the enzyme and its partial amino acid sequence (27 amino acids) was determined. A number of mouse and human expressed sequence tag clones were found to have high homology to the transcript of this partial amino acid sequence, and this information was utilized to clone a full length cDNA 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 carboxy terminus. The identity of the cloned enoyl CoA reductase cDNAs was confirmed by expressing the guinea pig and human cDNAs in E. 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 kb is present in most mouse tissues, with particularly high concentrations in liver and kidney.
J. Biol. Chem, 10.1074/jbc.M001168200
Submitted on February 10, 2000
Revised on April 20, 2000
Accepted on May 12, 2000
Molecular cloning and expression of mammalian peroxisomal trans 2-enoyl coenzyme a reductase cDNAs
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