2,4-Dienoyl-CoA Reductase from Escherichia coli Is a Novel Iron–Sulfur Flavoprotein That Functions in Fatty Acid -Oxidation Xiquan Liang,* Colin Thorpe,† and Horst Schulz* ,1 *Department of Chemistry, City College of the City University of New York, Convent Avenue at 138th Street, New York, New York 10031; and †Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Received April 12, 2000 2,4-Dienoyl-CoA reductase is an enzyme that is re- quired for the -oxidation of unsaturated fatty acids with even-numbered double bonds. The 2,4-dienoyl- CoA reductase from Escherichia coli was studied to explore the catalytic and structural properties that distinguish this enzyme from the corresponding eu- karyotic reductases. The E. coli reductase was found to contain 1 mol of flavin mononucleotide and 4 mol each of acid-labile iron and sulfur in addition to 1 mol of flavin adenine dinucleotide per mole of protein. Redox titrations revealed a requirement for 5 mol of electrons to completely reduce 1 mol of enzyme and provided evidence for the formation of a red semiqui- none intermediate. The reductase caused a significant polarization of the substrate carbonyl group as indi- cated by an enzyme-induced red shift of 38 nm in the spectrum of 5-phenyl-2,4-pentadienoyl-CoA. However, suspected cis 3 trans isomerase and  3 , 2 -enoyl-CoA isomerase activities were not detected in this enzyme. It is concluded that the 2,4-dienoyl-CoA reductases from E. coli and eukaryotic organisms are structurally and mechanistically unrelated enzymes that catalyze the same type of reaction with similar efficiencies. © 2000 Academic Press Key Words: 2,4-dienoyl-CoA reductase; E. coli; flavin mononucleotide; flavin adenine dinucleotide; iron– sulfur cluster. 2,4-Dienoyl-CoA reductase (EC 1.3.1.34) catalyzes the NADPH-dependent reduction of double bonds dur- ing the -oxidation of unsaturated fatty acids (for a review see Ref 1). Unsaturated fatty acid with even- numbered cis double bonds yield 2-trans,4-cis-dienoyl- CoA intermediates (2), whereas 2-trans,4-trans-dien- oyl-CoAs are formed from fatty acids with even-num- bered trans double bonds or odd-numbered cis double bonds (3). Surprisingly, 2,4-dienoyl-CoA reductase cat- alyzes the reduction of the 2-trans,4-cis and 2-trans,4- trans isomers with almost equal efficiency (4 – 6). This apparent absence of stereoselectivity is surprising and remains unexplained. It could be the consequence of a cis 3 trans isomerization catalyzed by the reductase so that the true substrate has an all trans configuration irrespective of the initial configuration of the 2,4-dien- oyl-CoA intermediate. The purifications of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli revealed profound differences between the mammalian and the bacterial enzymes (5–7). The former enzyme is a homotetramer with a native molecular mass of approximately 124 kDa, whereas the latter is a monomer with a mass of 73 kDa. Moreover, the bacterial reductase, in contrast to the mammalian enzyme, was reported to contain 1 mol of flavin adenine dinucleotide (FAD) 2 per mole of re- ductase. Molecular cloning of the rat (8, 9), human (10), mouse (11), and E. coli (12) reductases established that the mammalian enzymes are highly homologous to each other, but are dissimilar from the bacterial reduc- tase. Although the kinetic properties of the bovine and E. coli reductases are quite similar (4 – 6), their reac- tion products, 3-trans-enoyl-CoA (2, 5, 13) and 2-trans- enoyl-CoA (5, 7, 14) respectively, are different. This may reflect different reaction mechanisms or may be 1 To whom correspondence should be addressed. Fax: (212) 650- 8322. 2 Abbreviations used: FAD, flavin adenine dinucleotide; FMN, fla- vin mononucleotide; SDS–PAGE, sodium dodecyl sulfate–polyacryl- amide gel electrophoresis; TCA, trichloroacetic acid; IPTG, isopropyl -D-thiogalactoside. 0003-9861/00 $35.00 373 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 380, No. 2, August 15, pp. 373–379, 2000 doi:10.1006/abbi.2000.1941, available online at http://www.idealibrary.com on