Peroxisomes Contain Δ3,5,Δ2,4-Dienoyl-CoA Isomerase and thus Possess All Enzymes Required for the β-Oxidation of Unsaturated Fatty Acids by a Novel Reductase-Dependent Pathway

106

In Saccharomyces cerevisiae the metabolic degradation of saturated fatty acids is exclusively confined to peroxisomes. In addition to a functional ␤-oxidation system, the degradation of unsaturated fatty acids requires auxiliary enzymes, including a ⌬2,⌬3-enoyl-CoA isomerase and an NADPH-dependent 2,4-dienoyl-CoA reductase. We found both enzymes to be present in yeast peroxisomes. The impermeability of the peroxisomal membrane for pyrimidine nucleotides led to the question of how the NADPH needed by the reductase is regenerated in the peroxisomal lumen. We report the identification and functional analysis of the IDP3 gene product, which is a yeast peroxisomal NADP-dependent isocitrate dehydrogenase. The newly identified peroxisomal protein is homologous to the mitochondrial Idp1p and cytosolic Idp2p, which both are yeast NADPdependent isocitrate dehydrogenases. Yeast cells lacking Idp3p grow normally on saturated fatty acids, but growth is impaired on unsaturated fatty acids, indicating that the peroxisomal Idp3p is involved in their metabolic utilization. The data presented are consistent with the assumption that peroxisomes of S. cerevisiae contain the enzyme equipment needed for the degradation of unsaturated fatty acids, including an NADPdependent isocitrate dehydrogenase, a putative constituent of a peroxisomal NADPH-regenerating redox system.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

IDP3 Encodes a Peroxisomal NADP-dependent Isocitrate Dehydrogenase Required for the β-Oxidation of Unsaturated Fatty Acids

Henke

Girzalsky

Berteaux‐Lecellier

et al. 1998

106

“…1B) (7,14). The NADP ϩ -dependent pathway involves the sequential action of ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase, ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase, 2,4-dienoyl-CoA reductase, and, again, ⌬ 3 ,⌬ 2 -enoylCoA isomerase to yield 2-enoyl-CoA (9,10,15) (Fig. 1C).…”

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confidence: 97%

Molecular Characterization of Saccharomyces cerevisiae Δ3,Δ2-Enoyl-CoA Isomerase

Geisbrecht

Zhu

Schulz

et al. 1998

We report here the identification of the Saccharomyces cerevisiae peroxisomal ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase, an enzyme that is essential for the ␤-oxidation of unsaturated fatty acids. The yeast gene YLR284C was identified in an in silico screen for genes that contain an oleate response element, a transcription factor-binding site common to most fatty acid-induced genes. Growth on oleic acid resulted in a significant increase in YLR284C mRNA, demonstrating that it is indeed an oleate-induced gene. The deduced product of YLR284C contains a type 1 peroxisomal targeting signal-like sequence at its C terminus and localizes to the peroxisome in a PEX8-dependent manner. Removal of YLR284C from the S. cerevisiae genome eliminated growth on oleic acid, but had no effect on peroxisome biogenesis, indicating a role for YLR284C in fatty acid metabolism. Cells lacking YLR284C had no detectable ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase activity, and a bacterially expressed form of this protein catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 16 units/mg. We conclude that YLR284C encodes the yeast peroxisomal ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase and propose a new name, ECI1, to reflect its enoyl-CoA isomerase activity.

“…Thus, eukaryotic DCR requires another auxiliary enzyme, ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase, to convert trans-3-enoyl-CoA to trans-2-enoyl-CoA (1), which can be subsequently fed into the ␤-oxidation pathway. Lastly, eukaryotic DCR has been shown to metabolize fatty acids with double bonds at odd as well as even positions (5,6), whereas there is currently no evidence to support this for the E. coli enzyme.…”

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confidence: 99%

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

Hubbard

Liang

Schulz

et al. 2003

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...