A convenient assay for acyl-CoA-dehydrogenases

Dommes, Veronika; Kunau, Wolf‐H.

doi:10.1016/s0003-2697(76)80026-7

Cited by 75 publications

(57 citation statements)

References 6 publications

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“…Acyl-CoA dehydrogenases were determined according to Dommes and Kunau [18]. The method of Noyes and Bradshaw was employed to assay 3-hydroxyacyl-CoA dehydrogenase [19].…”

Section: Enzyme Assay5mentioning

confidence: 99%

Purification by Affinity Chromatography of 2,4-Dienoyl-CoA Reductases from Bovine Liver and Escherichia coli

et al. 1982

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Dye‐ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL‐6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2′,5′‐ADP (2′,5′‐ADP‐ Sepharose 4B) and immobilized 3′,5′‐ADP (3′,5′‐ADP‐ Agarose) were employed for purification of NADPH‐dependent 2‐enoyl‐CoA reductase and 2,4‐dienoyl‐CoA reductase from bovine liver (formerly called 4‐enoyl‐CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533–544], as well as 2,4‐dienoyl‐CoA reductase from Escherichia coli. The NADPH‐dependent 2‐enoyl‐CoA reductase from bovine liver mitochondria was separated from 2,4‐dienoyl‐CoA reductase by dye‐ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2′,5′‐ADP ‐ Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. The NADPH‐dependent 2,4‐dienoyl‐CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL‐6B, Matrex gel red A, and 2′,5′‐ADP–Sepharose 4B chromatography. The bacterial 2,4‐dienoyl‐CoA reductase was completely purified by ion‐exchange chromatography on DEAE‐cellulose followed by a single affinity chromatography step employing 2′,5′‐ADP ‐ Sepharose 4B and biospecific elution from the column with a substrate, trans, trans‐2,4‐decadienoyl‐CoA. The application of dye‐ligand and general ligand affinity chromatography for purification of NADPH‐dependent 2,4‐dienoyl‐CoA reductases taking part in the β‐oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.

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“…Acyl-CoA dehydrogenases were determined according to Dommes and Kunau [18]. The method of Noyes and Bradshaw was employed to assay 3-hydroxyacyl-CoA dehydrogenase [19].…”

Section: Enzyme Assay5mentioning

confidence: 99%

Purification by Affinity Chromatography of 2,4-Dienoyl-CoA Reductases from Bovine Liver and Escherichia coli

et al. 1982

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“…The Escherichia coli enzyme contains both FMN and FAD noncovalently bound to a single polypeptide, reducing the substrate by a hydride transfer mechanism in which reducing equivalents from NADPH are supplied indirectly to substrate (1). It functions as a monomer, having a molecular mass of 73 kDa, and has been shown recently to contain a 4Fe-4S cluster (4).…”

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The Crystal Structure and Reaction Mechanism of Escherichia coli 2,4-Dienoyl-CoA Reductase

Hubbard

Liang

Schulz

et al. 2003

Journal of Biological Chemistry

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Escherichia coli 2,4-dienoyl-CoA reductase is an ironsulfur flavoenzyme required for the metabolism of unsaturated fatty acids with double bonds at even carbon positions. The enzyme contains FMN, FAD, and a 4Fe-4S cluster and exhibits sequence homology to another ironsulfur flavoprotein, trimethylamine dehydrogenase. It also requires NADPH as an electron source, resulting in reduction of the C4-C5 double bond of the acyl chain of the CoA thioester substrate. The structure presented here of a ternary complex of E. coli 2,4-dienoyl-CoA reductase with NADP ؉ and a fatty acyl-CoA substrate reveals a possible mechanism for substrate reduction and provides details of a plausible electron transfer mechanism involving both flavins and the iron-sulfur cluster. The reaction is initiated by hydride transfer from NADPH to FAD, which in turn transfers electrons, one at a time, to FMN via the 4Fe-4S cluster. In the final stages of the reaction, the fully reduced FMN provides a hydride ion to the C5 atom of substrate, and Tyr-166 and His-252 are proposed to form a catalytic dyad that protonates the C4 atom of the substrate and complete the reaction. Inspection of the substrate binding pocket explains the relative promiscuity of the enzyme, catalyzing reduction of both 2-trans,4-cis-and 2-trans,4-trans-dienoyl-CoA thioesters.Metabolism of unsaturated fatty acids requires auxiliary enzymes in addition to those used in ␤-oxidation. After a given number of cycles through the ␤-oxidation pathway, those unsaturated fatty acyl-CoAs with double bonds at even-numbered carbon positions contain 2-trans,4-cis double bonds that cannot be modified by enoyl-CoA hydratase. Therefore, an auxiliary enzyme, 2,4-dienoyl-CoA reductase (DCR 1 ; EC 1.3.1.34), is used that utilizes NADPH to remove the C4-C5 double bond (1, 2). DCR is unusual in that it lacks stereospecificity, catalyzing the reduction of both natural fatty acids with cis double bonds, as well as substrates containing trans double bonds (3).Two structurally unrelated forms of DCR have been isolated, both of which use NADPH as reducing equivalents to catalyze reduction of a 2,4-dienoyl-CoA to an enoyl-CoA. The Escherichia coli enzyme contains both FMN and FAD noncovalently bound to a single polypeptide, reducing the substrate by a hydride transfer mechanism in which reducing equivalents from NADPH are supplied indirectly to substrate (1). It functions as a monomer, having a molecular mass of 73 kDa, and has been shown recently to contain a 4Fe-4S cluster (4). The enzyme produces 2-trans-enoyl-CoA (1), which can be incorporated into the ␤-oxidation pathway. In contrast, three homologous eukaryotic forms have been identified, two mitochondrial and one peroxisomal. Two of these lack flavin and iron-sulfur cofactors but are homotetramers with a total molecular mass of 124 kDa. (The third has not been characterized.) The substrate is reduced by a direct hydride transfer mechanism from NADPH to form trans-3-enoyl-CoA (1). Thus, eukaryotic DCR requires another auxiliary enzyme, ⌬ 3 ,⌬ 2 -enoy...

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“…In the last 10 years, it has become the subject of investigation in several laboratories. Enzyme from pig liver (Crane et al, 1956;Hall & Kamin, 1975), pig kidney , bovine liver (Dommes & Kunau, 1984;Murfin, 1974), bovine heart (Beinert, 1962;Davidson & Schultz, 1982), and rat liver (Ikeda et al, 1985a;Furuta et al, 1981) has been studied. Therefore, a certain variability can be found in the primary observations.…”

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“…Steady-state activity is also related to the acyl-CoA chain length (Hauge, 1956;Dommes & Kunau, 1984;Davidson & Schultz, 1982;Furuta et al, 1981;Hall et al, 1979;Ikeda et al, 198%) and can be correlated with the amplitude of this absorbance band (Hall et al, 1979;Ikeda et al, 1985~). This long-wavelength complex has been shown to be the donor for the physiological electron acceptor, electron-transferring flavoprotein (Hall & Lambeth, 1980;Gorelick et al, 1985).…”

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Oxidation-reduction of general acyl-CoA dehydrogenase by the butyryl-CoA/crotonyl-CoA couple. A new investigation of the rapid reaction kinetics

et al. 1988

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Pig kidney general acyl-CoA dehydrogenase (GAD) can be reduced by butyryl-CoA to form reduced enzyme and crotonyl-CoA. This reaction is reversible. Stopped-flow, kinetic investigations on GAD have been made, using the following reaction pairs: oxidized GAD/butyryl-CoA, oxidized GAD/crotonyl-CoA, oxidized GAD/cu,P-dideuteriobutyryl-CoA, reduced GAD/butyryl-CoA, and reduced GAD/ crotonyl-CoA (in 50 m M potassium phosphate buffer, pH 7.6 at 4 "C). Reduction of GAD by butyryl-CoA is triphasic. The slowest phase is 100-fold slower than the preceding phase and appears to represent a secondary process not directly related to the primary reduction events. The first two fast phases are responsible for reduction of GAD. Reduction proceeds via a reduced enzyme/crotonyl-CoA charge-transfer complex.a,P-Dideuteriobutyryl-CoA elicits a major deuterium isotope effect (1 5-fold) on the reduction reaction.Oxidation of G A D by crotonyl-CoA is biphasic. Oxidation proceeds via the same reduced enzyme/crotonyl-CoA charge-transfer complex seen during reduction. The oxidation reaction ends in a mixture composed largely of oxidized G A D species. From the data, we constructed a mechanism for the reduction/oxidation of G A D by butyryl-CoA/crotonyl-CoA. This mechanism was then used to simulate all of the observed kinetic time course data, using a single set of kinetic parameters. A close correspondence between the observed and simulated data was obtained.

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A convenient assay for acyl-CoA-dehydrogenases

Cited by 75 publications

References 6 publications

Purification by Affinity Chromatography of 2,4-Dienoyl-CoA Reductases from Bovine Liver and Escherichia coli

Purification by Affinity Chromatography of 2,4-Dienoyl-CoA Reductases from Bovine Liver and Escherichia coli

The Crystal Structure and Reaction Mechanism of Escherichia coli 2,4-Dienoyl-CoA Reductase

Oxidation-reduction of general acyl-CoA dehydrogenase by the butyryl-CoA/crotonyl-CoA couple. A new investigation of the rapid reaction kinetics

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