Purification and characterization of 2-enoyl-CoA reductase from bovine liver

Cvetanović, Miomir; Garza, M. M.; Dommes, Veronika; Kunau, Wolf‐H.

doi:10.1042/bj2270049

Cited by 14 publications

(7 citation statements)

References 55 publications

(52 reference statements)

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“…The mitochondrial chain elongation system is a reversal of the fatty acid ␤-oxidation system; thus, acetyl-CoA, instead of malonylCoA, is used for the condensation reaction, and the resulting ␤-keto acyl-CoA undergoes the same series of reactions as described above (3). The physiological function of the mitochondrial chain elongation system is not clear (4). Contradictory findings have been reported regarding the presence of another cellular fatty acid chain elongation system in mammalian peroxisomes.…”

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confidence: 80%

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Molecular Cloning and Expression of Mammalian Peroxisomaltrans-2-Enoyl-coenzyme A Reductase cDNAs

Das

Uhler

Hajra

2000

Journal of Biological Chemistry

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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 NADPHspecific 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.In mammalian cells, the fatty acid chain elongation system has been shown to be present in both endoplasmic reticulum (ER) 1 and mitochondria (1). The ER pathway is similar to the fatty acid synthesis system. In this multistep process, malonylCoA first condenses with acyl-CoA to form a ␤-keto acyl-CoA with two additional carbons with the release of CO 2 . The ␤-keto acyl-CoA then undergoes reduction of the ketone group followed by dehydration to form a 2-enoyl-CoA, which is then reduced by NADH or NADPH to form the longer (ϩ2C) acylCoA. Chain elongation and other biotransformation of fatty acids have been shown to occur primarily in the ER (1, 2). The mitochondrial chain elongation system is a reversal of the fatty acid ␤-oxidation system; thus, acetyl-CoA, instead of malonylCoA, is used for the condensation reaction, and the resulting ␤-keto acyl-CoA undergoes the same series of reactions as described above (3). The physiological function of the mitochondrial chain elongation system is not clear (4). Contradictory findings have been reported regarding the presence of another cellular fatty acid chain elongation system in mammalian peroxisomes. Nagi et al. (5) initially reported the absence of an elongation system in rat liver peroxisomes. However, Horie et al. (6) later provided good evidence for the presence of an active peroxisomal chain elongation system in ...

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confidence: 80%

“…Therefore, a separate 2-enoyl-CoA reductase is necessary for the reduction of the double bond at C-2 at the last step of elongation. Mitochondria have been shown to contain such a specific 2-enoyl-CoA reductase, which has been purified to homogeneity as a 35.5-kDa protein (4). This purified enzyme only utilized NADPH as the coenzyme.…”

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confidence: 99%

Molecular Cloning and Expression of Mammalian Peroxisomaltrans-2-Enoyl-coenzyme A Reductase cDNAs

Das

Uhler

Hajra

2000

Journal of Biological Chemistry

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“…Enoyl thioester reductases catalyze the reduction of the α,β-double bond of enoyl thioesters utilizing either NADH or NADPH as the electron donor (Figure ). Their typical physiological function is in primary metabolism, catalyzing the second reductive step in the cycles of either fatty acid elongation or de novo fatty acid biosynthesis. − These enzymes are therefore ubiquitous in nature and have been purified and characterized from both eucaryotes and procaryotes. − Enoyl thioester reductases are also implicated in certain secondary metabolic processes, in particular formation of the macrolide and polyether polyketide antibiotics. − …”

Section: Introductionmentioning

confidence: 99%

“…[1][2][3][4][5] These enzymes are therefore ubiquitous in nature and have been purified and characterized from both eucaryotes and procaryotes. [6][7][8][9][10] Enoyl thioester reductases are also implicated in certain secondary metabolic processes, in particular formation of the macrolide and polyether polyketide antibiotics. [11][12][13][14][15][16] Extensive regiochemical and stereochemical studies have been reported for enoyl thioester reductases.…”

Section: Introductionmentioning

confidence: 99%

Linking Diversity in Evolutionary Origin and Stereospecificity for Enoyl Thioester Reductases: Determination and Interpretation of the Novel Stereochemical Course of Reaction Catalyzed by Crotonyl CoA Reductase fromStreptomyces collinus

1997

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The stereochemical course of reduction of crotonyl CoA by the novel crotonyl CoA reductase (CCR) of Streptomyces collinus was determined using a radiochemical assay. The reaction was shown to proceed with transfer of the hydrogen from the pro-4S position of NADPH to the Re face of the β-carbon of crotonyl CoA. This transfer represents the first exception to the observation that enoyl thioester reductases catalyze transfer of the pro-4S hydrogen of NADPH to the Si face of the substrate. The observation of addition of solvent hydrogen to the Re face of the R-carbon in the reaction catalyzed by CCR demonstrated that the overall reduction of crotonyl CoA proceeds in an anti fashion. The overall stereochemical outcome of the reaction catalyzed by CCR is different to the four stereochemical outcomes that have previously been observed for enoyl thioester reductases. It is significant that the predicted amino acid sequence of CCR has also been shown to be unrelated to other enoyl thioester reductases. Based on these observations it is proposed that the stereochemical course of an enoyl thioester reduction serves no mechanistic function but merely reflects the pedigree or ancestral lineage of the enzyme. Any two enoyl thioester reductases which exhibit different stereospecificities are, therefore, predicted to have different pedigrees and unrelated amino acid sequences. An evaluation of all the enoyl thioester reductases where both the stereochemical course of reduction and the predicted amino acid sequence are known is shown to be entirely consistent with these predictions.

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“…These results point to the role of thiol groups in the activity of these enzymes. 1-Cyclohexenylcarbonyl-CoA reductase, like other enoyl-CoA reductases, is inhibited by divalent cations only (if preincubated without cofactor), by high concentrations of NADPH, and by the enzymatic products NADP and cyclohexylcarbonyl-CoA (6,13,17,21,29). The enzyme appears to have no requirement for flavin adenine dinucleotide or flavin mononucleotide (in contrast to a trans-2-enoyl-CoA reductase from Euglena gracilis [13] or Mycobactenium smegmatis [17]) and to specifically require the CoA thioester.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus

et al. 1992

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A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces coUinus. This enzyme, a dimer with subunits of identical Mr (36,000), exhibits a Km of 1.5 0.3 ,uM for NADPH and 25 + 3 ,uM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30°C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained.

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Purification and characterization of 2-enoyl-CoA reductase from bovine liver

Cited by 14 publications

References 55 publications

Molecular Cloning and Expression of Mammalian Peroxisomaltrans-2-Enoyl-coenzyme A Reductase cDNAs

Molecular Cloning and Expression of Mammalian Peroxisomaltrans-2-Enoyl-coenzyme A Reductase cDNAs

Linking Diversity in Evolutionary Origin and Stereospecificity for Enoyl Thioester Reductases: Determination and Interpretation of the Novel Stereochemical Course of Reaction Catalyzed by Crotonyl CoA Reductase fromStreptomyces collinus

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus

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