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Originally published In Press as doi:10.1074/jbc.M001168200 on May 12, 2000

J. Biol. Chem., Vol. 275, Issue 32, 24333-24340, August 11, 2000
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Molecular Cloning and Expression of Mammalian Peroxisomal trans-2-Enoyl-coenzyme A Reductase cDNAs*

Arun K. Das, Michael D. Uhler, and Amiya K. HajraDagger

From the Mental Health Research Institute and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104-1687

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.

* This work was supported by National Institutes of Health Grant NS 15747.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF232009, AF232010, and AF232011.

Dagger To whom correspondence should be addressed: Neuroscience Laboratory, University of Michigan, 1103 E. Huron St., Ann Arbor, MI 48104-1687. Tel.: 734-763-4368; Fax: 734-936-2690; E-mail: akhajra@umich.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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