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J. Biol. Chem., Vol. 280, Issue 6, 4329-4338, February 11, 2005

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Mitochondrial trans-2-Enoyl-CoA Reductase of Wax Ester Fermentation from Euglena gracilis Defines a New Family of Enzymes Involved in Lipid Synthesis^*

Meike Hoffmeister, Markus Piotrowski, Ulrich Nowitzki, and William Martin¶

From the Institute of Botany III, University of Düsseldorf, D-40225 Düsseldorf, Germany and the Department of Plant Physiology, University of Bochum, D-44801 Bochum, Germany

Under anaerobiosis, Euglena gracilis mitochondria perform a malonyl-CoA independent synthesis of fatty acids leading to accumulation of wax esters, which serve as the sink for electrons stemming from glycolytic ATP synthesis and pyruvate oxidation. An important enzyme of this unusual pathway is trans-2-enoyl-CoA reductase (EC 1.3.1.44), which catalyzes reduction of enoyl-CoA to acyl-CoA. Trans-2-enoyl-CoA reductase from Euglena was purified 1700-fold to electrophoretic homogeneity and was active with NADH and NADPH as the electron donor. The active enzyme is a monomer with molecular mass of 44 kDa. The amino acid sequence of tryptic peptides determined by electrospray ionization mass spectrometry were used to clone the corresponding cDNA, which encoded a polypeptide that, when expressed in Escherichia coli and purified by affinity chromatography, possessed trans-2-enoyl-CoA reductase activity close to that of the enzyme purified from Euglena. Trans-2-enoyl-CoA reductase activity is present in mitochondria and the mRNA is expressed under aerobic and anaerobic conditions. Using NADH, the recombinant enzyme accepted crotonyl-CoA (k_m = 68 µM) and trans-2-hexenoyl-CoA (k_m = 91 µM). In the crotonyl-CoA-dependent reaction, both NADH (k_m = 109 µM) or NADPH (k_m = 119 µM) were accepted, with 2–3-fold higher specific activities for NADH relative to NADPH. Trans-2-enoyl-CoA reductase homologues were not found among other eukaryotes, but are present as hypothetical reading frames of unknown function in sequenced genomes of many proteobacteria and a few Gram-positive eubacteria, where they occasionally occur next to genes involved in fatty acid and polyketide biosynthesis. Trans-2-enoyl-CoA reductase assigns a biochemical activity, NAD(P)H-dependent acyl-CoA synthesis from enoyl-CoA, to one member of this gene family of previously unknown function.

Received for publication, September 24, 2004 , and in revised form, November 24, 2004.

^* This work was supported by the Deutsche Forschungsgemeinschaft and by BASF Plant Science. The costs of publication of this article were defrayed in part by the payment of page charges. This 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 GenBank^TM/EBI Data Bank with accession number(s) AY741582.

^¶ To whom correspondence should be addressed: Institute of Botany III, University of Düsseldorf, D-40225 Düsseldorf, Germany. Tel.: 49-211-811-3011; Fax: 49-211-811-3554; E-mail: w.martin{at}uni-duesseldorf.de.

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