GO:0008670 2,4-dienoyl-CoA reductase (NADPH) activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0008670 describes the NADPH-dependent reduction of 2,4-dienoyl-CoA to a 4,5-saturated enoyl-CoA, a reaction required for the complete beta-oxidation of unsaturated fatty acids.
• The reaction proceeds stepwise through a dienolate intermediate, as shown by kinetic and spectroscopic studies of the enzyme.
• The enzyme exists in mitochondrial and peroxisomal isoforms encoded by distinct genes, including DECR1 in mammals and SPS19 in yeast.
• Loss or deficiency of 2,4-dienoyl-CoA reductase impairs fatty acid oxidation and has been linked to metabolic and pathophysiological conditions.
• The activity is assayed radiochemically or spectrophotometrically by following NADPH oxidation or product formation.
• CRISPR-based knockout, knock-in, and overexpression models enable causal dissection of DECR1 and related genes in fatty acid oxidation and disease.
Description
GO:0008670, 2,4-dienoyl-CoA reductase (NADPH) activity, is a molecular function that catalyzes the NADPH-dependent reduction of 2,4-dienoyl-CoA substrates to 4,5-saturated enoyl-CoA products. This activity is essential for the beta-oxidation of unsaturated fatty acids, because the standard beta-oxidation cycle cannot directly process double bonds at even-numbered positions without auxiliary enzymes. The reaction is reversible and involves both (2E,4E)- and (2E,4Z)-dienoyl-CoA isomers, with NADP+ and NADPH as the redox couple. Researchers study this activity because it sits at the intersection of mitochondrial and peroxisomal fatty acid oxidation, and because its dysfunction is associated with impaired energy metabolism and pathophysiological states. The enzyme has been purified and characterized from rat liver mitochondria, mouse peroxisomes, and human mitochondria, enabling detailed mechanistic and structural work. Yeast genetics identified the oleate-inducible SPS19 gene as encoding the peroxisomal 2,4-dienoyl-CoA reductase, providing a tractable model for functional studies. Methodologically, the activity can be measured by radioactive assays that quantify product formation from labeled substrates, or by spectrophotometric monitoring of NADPH consumption. These assays, combined with modern CRISPR models, allow precise interrogation of the enzyme's role in fatty acid oxidation and metabolic disease.
2,4-dienoyl-CoA reductase (NADPH) activity At A Glance
| GO ID | GO:0008670 |
|---|---|
| GO term | 2,4-dienoyl-CoA reductase (NADPH) activity |
| Ontology | molecular_function |
| Synonym | 4-enoyl-CoA reductase activity; 4-enoyl-CoA reductase (NADPH2); 4-enoyl-CoA reductase (NADPH) activity; 4-enoyl coenzyme A (reduced nicotinamide adenine dinucleotide phosphate) reductase activity; trans-2,3-didehydroacyl-CoA:NADP+ 4-oxidoreductase activity |
| Major function | NADPH-dependent reduction of 2,4-dienoyl-CoA to 4,5-saturated enoyl-CoA, supporting unsaturated fatty acid beta-oxidation |
| Substrates | (2E,4E)-dienoyl-CoA and (2E,4Z)-dienoyl-CoA |
| Cofactor | NADPH / NADP+ |
| Cellular locations | Mitochondria and peroxisomes |
| Representative genes | DECR1 (mammals), SPS19 (Saccharomyces cerevisiae), PDCR (mouse peroxisomal) |
What Is GO:0008670?
2,4-dienoyl-CoA reductase (NADPH) activity is the catalysis of two interconverting reactions: a 4,5-saturated-(2E)-enoyl-CoA plus NADP+ yields a (2E,4E)-dienoyl-CoA plus H+ and NADPH, and a (2E,4Z)-dienoyl-CoA plus H+ and NADPH yields a 4,5-saturated-(2E)-enoyl-CoA plus NADP+. In other words, the enzyme uses NADPH to reduce a conjugated dienoyl-CoA to a saturated enoyl-CoA, and can also operate in the reverse direction with NADP+. This activity is also known as 4-enoyl-CoA reductase activity or trans-2,3-didehydroacyl-CoA:NADP+ 4-oxidoreductase activity.
Why Is 2,4-dienoyl-CoA reductase (NADPH) activity Important in Cell Biology?
This activity is critical for completing the beta-oxidation of unsaturated fatty acids, which cannot be fully degraded by the core beta-oxidation spiral alone. By reducing 2,4-dienoyl-CoA intermediates, the enzyme prevents the accumulation of unsaturated acyl-CoA species that would otherwise stall fatty acid oxidation. Its importance extends to energy homeostasis, because impaired fatty acid oxidation affects ATP production and metabolic flux. The enzyme has been characterized in mitochondria and peroxisomes, and its isoforms are encoded by distinct genes, making it a model for compartmentalized lipid metabolism. Clinically, altered 2,4-dienoyl-CoA reductase activity has been discussed in the context of pathophysiological conditions, motivating further research.
• Enables beta-oxidation of unsaturated fatty acids by removing double bonds that block the standard cycle.
• Uses NADPH as a cofactor, linking fatty acid oxidation to cellular redox balance.
• Exists in mitochondrial and peroxisomal compartments, supporting compartment-specific lipid metabolism.
• The reaction proceeds via a dienolate intermediate, making it mechanistically distinct from simple hydride-transfer dehydrogenases.
• Deficiency or dysregulation is associated with impaired fatty acid oxidation and pathophysiological states.
• Growth hormone increases mitochondrial 2,4-dienoyl-CoA reductase activity, indicating hormonal regulation of the enzyme.
• The yeast SPS19 gene is oleate-inducible, linking the activity to lipid-responsive transcriptional programs.
• Assays for the activity support diagnosis and mechanistic studies of fatty acid oxidation disorders.
• The enzyme is a target for structural and kinetic studies of dienoyl-CoA reduction.
• CRISPR models enable causal testing of gene function in metabolic and disease contexts.
Molecular Mechanism of 2,4-dienoyl-CoA reductase (NADPH) activity
Substrate recognition and binding
In simple terms: The enzyme first grabs the dienoyl-CoA molecule and holds it in place.
2,4-dienoyl-CoA reductase binds conjugated dienoyl-CoA substrates, including (2E,4E)- and (2E,4Z)-dienoyl-CoA, positioning the diene for reduction. The CoA moiety anchors the substrate, while the diene portion is oriented toward the NADPH cofactor. This binding mode ensures specificity for 2,4-dienoyl-CoA over saturated acyl-CoA species.
Hydride transfer and dienolate intermediate
In simple terms: NADPH hands over a hydride, creating a short-lived intermediate before the final product forms.
The reduction proceeds stepwise: NADPH transfers a hydride to the dienoyl-CoA, generating a dienolate intermediate that has been observed experimentally. This intermediate then undergoes protonation to yield the 4,5-saturated enoyl-CoA product. The stepwise mechanism distinguishes this enzyme from concerted reductases and explains its stereochemical outcomes.
Cofactor specificity and redox chemistry
In simple terms: The enzyme uses NADPH, not NADH, as its preferred reducing agent.
The reaction is NADPH-dependent, with NADP+ and NADPH forming the redox couple. The enzyme can also catalyze the reverse reaction, oxidizing a 4,5-saturated-(2E)-enoyl-CoA to a (2E,4E)-dienoyl-CoA while reducing NADP+. This reversibility may allow the enzyme to buffer dienoyl-CoA levels under different metabolic conditions.
Isoforms and subcellular localization
In simple terms: Different versions of the enzyme work in different parts of the cell.
Mammals express mitochondrial and peroxisomal isoforms of 2,4-dienoyl-CoA reductase, encoded by distinct genes such as DECR1 and PDCR. The yeast peroxisomal enzyme is encoded by the oleate-inducible SPS19 gene. These isoforms share catalytic activity but differ in localization and regulation, allowing fatty acid oxidation to proceed in multiple compartments.
Regulation by hormonal and nutritional signals
In simple terms: Hormones and diet can change how much of this enzyme activity is available.
Growth hormone increases mitochondrial 2,4-dienoyl-CoA reductase activity, indicating endocrine control of the enzyme. In yeast, SPS19 is induced by oleate, linking the activity to lipid availability. Such regulation helps match fatty acid oxidation capacity to metabolic demand.
Key Genes Involved in GO:0008670 2,4-dienoyl-CoA reductase (NADPH) activity
The following genes and proteins are directly associated with 2,4-dienoyl-CoA reductase (NADPH) activity or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| DECR1 | Mitochondrial 2,4-dienoyl-CoA reductase in mammals | Core enzyme for unsaturated fatty acid beta-oxidation; target for metabolic studies |
| PDCR | Mouse peroxisomal 2,4-dienoyl-CoA reductase | Model for peroxisomal fatty acid oxidation |
| SPS19 | Yeast peroxisomal 2,4-dienoyl-CoA reductase | Oleate-inducible model for lipid metabolism |
| NADP+ | Oxidized cofactor | Redox partner in the reverse reaction |
| NADPH | Reduced cofactor | Provides reducing equivalents for dienoyl-CoA reduction |
| Acyl-CoA dehydrogenases | Generate enoyl-CoA intermediates | Upstream of dienoyl-CoA reductase in beta-oxidation |
| Enoyl-CoA hydratase | Hydrates enoyl-CoA | Downstream enzyme in the beta-oxidation cycle |
| 3-hydroxyacyl-CoA dehydrogenase | Oxidizes hydroxyacyl-CoA | Beta-oxidation cycle component |
| 3-ketoacyl-CoA thiolase | Cleaves ketoacyl-CoA | Completes beta-oxidation cycle |
| CPT1 | Mitochondrial fatty acid import | Upstream regulator of fatty acid oxidation flux |
| PPARalpha | Transcriptional regulator of lipid metabolism | Controls expression of fatty acid oxidation genes |
| Growth hormone | Hormonal regulator | Increases mitochondrial 2,4-dienoyl-CoA reductase activity |
| ACOX1 | Peroxisomal acyl-CoA oxidase | Generates substrates for peroxisomal beta-oxidation |
| HADHA | Mitochondrial trifunctional protein subunit | Coordinates beta-oxidation steps |
| HADHB | Mitochondrial trifunctional protein subunit | Coordinates beta-oxidation steps |
| SLC25A17 | Peroxisomal CoA transporter | Supports peroxisomal beta-oxidation |
| PEX5 | Peroxisomal import receptor | Required for peroxisomal enzyme localization |
How Is 2,4-dienoyl-CoA reductase (NADPH) activity Regulated?
2,4-dienoyl-CoA reductase (NADPH) activity is regulated at multiple levels. Hormonally, growth hormone increases mitochondrial enzyme activity, linking it to endocrine control of fatty acid oxidation. Nutritionally, the yeast SPS19 gene is induced by oleate, indicating that lipid availability drives expression of the peroxisomal enzyme. At the protein level, the enzyme requires NADPH as a cofactor, so cellular redox status influences flux through the reaction. Compartmentalization into mitochondria and peroxisomes provides additional regulatory separation, with distinct genes encoding the isoforms. These layers of control help match dienoyl-CoA reduction to the overall rate of fatty acid oxidation.
2,4-dienoyl-CoA reductase (NADPH) activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| DECR1 | Impaired unsaturated fatty acid beta-oxidation | CRISPR knockout in human cell lines; metabolic flux assays |
| PDCR | Peroxisomal fatty acid oxidation defects | Mouse knockout; peroxisomal substrate oxidation assays |
| SPS19 | Yeast lipid metabolism and oleate response | Yeast deletion and overexpression; growth on oleate |
| Growth hormone pathway | Endocrine regulation of fatty acid oxidation | Hypophysectomy models; hormone supplementation |
| NADPH redox balance | Metabolic stress and redox homeostasis | CRISPR knock-in of redox-sensitive reporters |
Fatty acid oxidation disorders
Deficiency or dysfunction of 2,4-dienoyl-CoA reductase impairs the beta-oxidation of unsaturated fatty acids, which can contribute to metabolic decompensation under conditions of high fatty acid flux. Because the enzyme is required to remove double bonds that block the beta-oxidation spiral, its loss may lead to accumulation of unsaturated acyl-CoA intermediates. Research has discussed the pathophysiological significance of 2,4-dienoyl-CoA reductases in metabolic disease.
Metabolic and endocrine conditions
Growth hormone increases mitochondrial 2,4-dienoyl-CoA reductase activity, suggesting that endocrine states affecting growth hormone levels may alter fatty acid oxidation capacity. This links the enzyme to broader metabolic regulation and energy homeostasis. Further studies are needed to define how changes in activity contribute to specific clinical phenotypes.
Peroxisomal disorders
The peroxisomal isoform of 2,4-dienoyl-CoA reductase, encoded by PDCR in mouse and SPS19 in yeast, participates in peroxisomal fatty acid oxidation. Disruption of peroxisomal function could therefore affect the processing of unsaturated fatty acids. Model organisms have been valuable for dissecting these pathways.
From 2,4-dienoyl-CoA reductase (NADPH) activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of DECR1 impair unsaturated fatty acid oxidation? | CRISPR knockout in HepG2 or HEK293 cells |
| Does a point mutation in the active site abolish catalytic activity? | CRISPR point mutation knock-in in DECR1 |
| Can a tagged DECR1 be used to monitor localization? | Knock-in of fluorescent or affinity tag |
| Does overexpression of DECR1 increase fatty acid oxidation flux? | CRISPR overexpression or cDNA overexpression |
| Is SPS19 required for growth on oleate? | Yeast SPS19 deletion and overexpression |
| Does PDCR loss affect peroxisomal beta-oxidation? | Mouse PDCR knockout |
How to Study the 2,4-dienoyl-CoA reductase (NADPH) activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Radioactive assay | Product formation from labeled dienoyl-CoA | Enzyme activity in tissues or purified fractions |
| NADPH oxidation assay | Decrease in NADPH absorbance | Kinetic characterization and inhibitor testing |
| Recombinant expression | Purified enzyme for in vitro studies | Structural and mechanistic analysis |
| Yeast genetics | Growth on oleate and enzyme activity | Functional dissection of SPS19 |
| CRISPR knockout | Loss-of-function phenotype | Causal testing of DECR1 in cells |
| CRISPR knock-in | Tagged or mutant enzyme | Localization and activity studies |
| Metabolic flux analysis | Fatty acid oxidation rate | Pathway-level consequences of gene manipulation |
| Transcriptomics | Expression of lipid metabolism genes | Regulatory network analysis |
Radioactive assays for enzyme activity
Radioactive assays using labeled dienoyl-CoA substrates allow direct quantification of 2,4-dienoyl-CoA reductase activity by measuring product formation. These assays are sensitive and can be applied to tissue homogenates or purified enzyme preparations. They are useful for validating enzyme function after genetic manipulation.
Spectrophotometric NADPH assays
Because the reaction consumes NADPH, enzyme activity can be monitored spectrophotometrically by following the decrease in absorbance at 340 nm. This method is convenient for kinetic studies and inhibitor screening. It can also be adapted to measure the reverse reaction using NADP+.
Recombinant protein expression and purification
Recombinant 2,4-dienoyl-CoA reductase has been cloned, expressed, and purified from rat liver mitochondria, mouse peroxisomes, and human mitochondria. His-tagged human mitochondrial enzyme enables rapid purification for structural and kinetic studies. These reagents support detailed mechanistic work.
Genetic and CRISPR models
CRISPR knockout, point mutation, knock-in, and overexpression models allow causal testing of gene function in fatty acid oxidation. Yeast genetics with SPS19 provides a complementary system for pathway dissection. Combining these models with metabolic assays links genotype to flux.
How CRISPR Can Be Used to Study GO:0008670 2,4-dienoyl-CoA reductase (NADPH) activity
Knockout
CRISPR knockout of DECR1 or PDCR eliminates 2,4-dienoyl-CoA reductase activity, allowing researchers to test its requirement for unsaturated fatty acid beta-oxidation. Knockout cells can be challenged with unsaturated fatty acids to reveal metabolic vulnerabilities. Such models are essential for distinguishing the enzyme's role from that of other beta-oxidation enzymes.
Point Mutation
CRISPR point mutation can introduce catalytic dead or substrate-binding mutations into DECR1, enabling separation of enzymatic activity from other protein functions. These models help validate the stepwise mechanism involving the dienolate intermediate. They also allow testing of disease-associated variants.
Knock-in
Knock-in of fluorescent or affinity tags into the endogenous DECR1 locus enables real-time localization and interaction studies. Tagged knock-in models preserve native regulation while providing tools for imaging and proteomics. They are valuable for studying mitochondrial and peroxisomal targeting.
Overexpression
CRISPR-mediated overexpression or cDNA overexpression of DECR1 increases enzyme levels, allowing researchers to test whether elevated activity enhances fatty acid oxidation flux. Overexpression models can also reveal dose-dependent effects on redox balance. They complement loss-of-function studies for bidirectional causal inference.
How EDITGENE Supports 2,4-dienoyl-CoA reductase (NADPH) activity Research
Researchers studying 2,4-dienoyl-CoA reductase (NADPH) activity-related genes often need to determine whether a candidate gene is causally involved in fatty acid oxidation, metabolic regulation, or disease. EDITGENE provides CRISPR-based cell model services that enable precise genetic manipulation of DECR1, PDCR, SPS19, and related genes, from knockout to knock-in and overexpression.
Contact EDITGENE today to design your custom CRISPR model for 2,4-dienoyl-CoA reductase (NADPH) activity research.
Frequently Asked Questions About 2,4-dienoyl-CoA reductase (NADPH) activity
What is 2,4-dienoyl-CoA reductase (NADPH) activity?
It is a molecular function (GO:0008670) that catalyzes the NADPH-dependent reduction of 2,4-dienoyl-CoA to a 4,5-saturated enoyl-CoA, supporting unsaturated fatty acid beta-oxidation.
What genes are involved in 2,4-dienoyl-CoA reductase (NADPH) activity?
Key genes include DECR1 in mammals, PDCR in mouse peroxisomes, and SPS19 in yeast.
Where does 2,4-dienoyl-CoA reductase (NADPH) activity occur in the cell?
The activity occurs in mitochondria and peroxisomes, with distinct isoforms encoded by different genes.
What is the mechanism of 2,4-dienoyl-CoA reductase?
The reaction proceeds stepwise through a dienolate intermediate, with NADPH providing a hydride to the dienoyl-CoA substrate.
Why is 2,4-dienoyl-CoA reductase important for fatty acid oxidation?
It removes double bonds that block the beta-oxidation spiral, allowing complete oxidation of unsaturated fatty acids.
How is 2,4-dienoyl-CoA reductase activity measured?
It can be measured by radioactive assays or by spectrophotometric monitoring of NADPH oxidation.
Is 2,4-dienoyl-CoA reductase regulated by hormones?
Yes, growth hormone increases mitochondrial 2,4-dienoyl-CoA reductase activity.
What diseases are linked to 2,4-dienoyl-CoA reductase deficiency?
Deficiency has been discussed in the context of impaired fatty acid oxidation and pathophysiological conditions.
Can CRISPR be used to study 2,4-dienoyl-CoA reductase?
Yes, CRISPR knockout, point mutation, knock-in, and overexpression models enable causal studies of DECR1 and related genes.
What is the yeast homolog of 2,4-dienoyl-CoA reductase?
The Saccharomyces cerevisiae peroxisomal enzyme is encoded by the oleate-inducible SPS19 gene.
Conclusion
GO:0008670, 2,4-dienoyl-CoA reductase (NADPH) activity, is a mechanistically distinctive molecular function required for unsaturated fatty acid beta-oxidation. Its stepwise dienolate mechanism, NADPH dependence, and dual mitochondrial/peroxisomal localization make it a rich subject for metabolic research. CRISPR-based cell models now allow precise causal interrogation of DECR1, PDCR, SPS19, and related genes, linking genotype to fatty acid oxidation flux and disease-relevant phenotypes. EDITGENE provides the full spectrum of knockout, point mutation, knock-in, overexpression, and screening services to accelerate this research.
References
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- 2. Kimura C et al.. 2004. [2,4-Dienoyl-CoA reductases: from discovery toward pathophysiological significance].. Nihon Rinsho 62(8):1577-83 PMID: 15344554
- 3. Fillgrove KL et al.. 1999. Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria.. Protein Expr Purif 17(1):57-63 PMID: 10497069
- 4. Nada MA et al.. 1992. Radioactive assay of 2,4-dienoyl-coenzyme A reductase.. Anal Biochem 201(1):62-7 PMID: 1621963
- 5. Gurvitz A et al.. 1997. The Saccharomyces cerevisiae peroxisomal 2,4-dienoyl-CoA reductase is encoded by the oleate-inducible gene SPS19.. J Biol Chem 272(35):22140-7 PMID: 9268358
- 6. Geisbrecht BV et al.. 1999. The mouse gene PDCR encodes a peroxisomal delta(2), delta(4)-dienoyl-CoA reductase.. J Biol Chem 274(36):25814-20 PMID: 10464321
- 7. Chu X et al.. 2003. Expression, purification, and characterization of His-tagged human mitochondrial 2,4-dienoyl-CoA reductase.. Protein Expr Purif 31(2):292-7 PMID: 14550650
- 8. Clejan S et al.. 1986. Effect of growth hormone on fatty acid oxidation: growth hormone increases the activity of 2,4-dienoyl-CoA reductase in mitochondria.. Arch Biochem Biophys 246(2):820-8 PMID: 3707134
