GO:0080023 (2E)-enoyl-CoA hydratase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0080023 describes (2E)-enoyl-CoA hydratase activity, the reversible hydration of a (2E)-enoyl-CoA to a (3R)-3-hydroxyacyl-CoA.
• The reaction is a core step of fatty acid beta-oxidation and is also required for bile acid synthesis in peroxisomes.
• The enzyme is found in monofunctional and multifunctional forms, including peroxisomal MFE2 and mitochondrial MFE1.
• Structural enzymology has shown that the active site and substrate channeling control catalysis in trifunctional and multifunctional enzymes.
• The enzyme can act on non-CoA enoyl-thioesters when the oxyanion hole is activated by 3',5'-adenosine-diphosphate.
• Loss or dysfunction of this activity is linked to defects in fatty acid oxidation and peroxisomal metabolism.
Description
GO:0080023, (2E)-enoyl-CoA hydratase activity, is a molecular function that catalyzes the reversible conversion of a (2E)-enoyl-CoA to a (3R)-3-hydroxyacyl-CoA and water. This activity is central to fatty acid beta-oxidation, where it prepares the acyl-CoA chain for subsequent dehydrogenation and thiolysis. In peroxisomes, the same activity participates in the degradation of unsaturated fatty acids and in bile acid synthesis. Researchers study this term because it defines a key enzymatic step in lipid catabolism and because its dysfunction can disrupt energy homeostasis and peroxisomal metabolism. The activity is carried out by monofunctional enzymes and by multifunctional enzymes that combine hydratase, dehydrogenase, and thiolase activities. Structural and enzymological studies continue to reveal how substrate specificity, active-site geometry, and conformational flexibility shape catalysis.
(2E)-enoyl-CoA hydratase activity At A Glance
| GO ID | GO:0080023 |
|---|---|
| GO term | (2E)-enoyl-CoA hydratase activity |
| Ontology | molecular_function |
| Synonym | (3R)-3-hydroxyacyl-CoA dehydratase activity; 3R-hydroxyacyl-CoA dehydratase activity |
| Definition | Catalysis of the reaction: a (3R)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O. |
| Major function | Hydration/dehydration of enoyl-CoA and hydroxyacyl-CoA intermediates in fatty acid oxidation |
| Pathway context | Fatty acid beta-oxidation; peroxisomal fatty acid degradation; bile acid synthesis |
| Representative enzymes | Peroxisomal multifunctional enzyme 2 (MFE2); monofunctional peroxisomal enoyl-CoA hydratase 2; mitochondrial multifunctional enzyme 1 (MFE1); bacterial trifunctional enzyme |
What Is GO:0080023?
According to the QuickGO definition, GO:0080023 is the catalysis of the reaction: a (3R)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O. In other words, it is the reversible removal or addition of water across the double bond of a (2E)-enoyl-CoA to produce a (3R)-3-hydroxyacyl-CoA. The term is also known as (3R)-3-hydroxyacyl-CoA dehydratase activity or 3R-hydroxyacyl-CoA dehydratase activity.
Why Is (2E)-enoyl-CoA hydratase activity Important in Cell Biology?
GO:0080023 is important because it defines a rate-contributing step in fatty acid beta-oxidation and related lipid pathways. The reaction allows the carbon chain to be further oxidized, and its correct stereochemistry is essential for downstream dehydrogenase and thiolase steps. In peroxisomes, this activity is required for the breakdown of specific unsaturated fatty acids and for bile acid synthesis. Because the same activity can be embedded in multifunctional enzymes, it also serves as a model for understanding substrate channeling and conformational regulation in metabolic complexes.
• Catalyzes a central step in fatty acid beta-oxidation.
• Participates in peroxisomal degradation of even cis-unsaturated fatty acids.
• Contributes to bile acid synthesis in peroxisomes.
• Provides stereospecific (3R)-hydroxyacyl-CoA intermediates for downstream enzymes.
• Is embedded in multifunctional enzymes that coordinate multiple beta-oxidation steps.
• Is a target for structural enzymology studies of substrate specificity and active-site chemistry.
• Can act on non-CoA enoyl-thioesters under specific activation conditions.
• Its dysfunction is relevant to inherited disorders of fatty acid and peroxisomal metabolism.
Molecular Mechanism of (2E)-enoyl-CoA hydratase activity
Substrate binding and recognition
In simple terms: The enzyme first grabs the enoyl-CoA substrate and positions it correctly.
The enzyme binds a (2E)-enoyl-CoA substrate in an elongated conformation that places the double bond near the catalytic residues. Structural studies of the Mycobacterium tuberculosis trifunctional enzyme show that substrate specificity is influenced by the shape and flexibility of the binding pocket. In multifunctional enzyme type-1 (MFE1), the substrate 2E-decenoyl-CoA and 2E,4E-decadienoyl-CoA occupy a channel that connects the hydratase and dehydrogenase sites.
Catalytic hydration/dehydration
In simple terms: Water is added or removed across the double bond to convert the substrate back and forth.
The catalytic reaction is the reversible addition of water to the (2E)-enoyl-CoA to form a (3R)-3-hydroxyacyl-CoA. The reaction is stereospecific and produces the (3R)-hydroxyacyl-CoA intermediate required for beta-oxidation. In the peroxisomal multifunctional enzyme 2, the hydratase reaction is essential for bile acid synthesis.
Active-site oxyanion hole and cofactor effects
In simple terms: A special pocket in the enzyme stabilizes the reaction intermediate and can be switched on by a small molecule.
The active site contains an oxyanion hole that stabilizes the transition state during hydration. Recent work shows that 3',5'-adenosine-diphosphate can activate the oxyanion hole and enable competent catalysis on non-CoA enoyl-thioesters. This finding highlights how cofactor-like molecules can modulate the catalytic competence of enoyl-CoA hydratases.
Substrate channeling in multifunctional enzymes
In simple terms: In large enzymes, the product of one reaction is passed directly to the next active site.
In multifunctional enzyme type-1 (MFE1), the hydratase and dehydrogenase activities are coupled, and the regeneration of the dehydrogenase catalytic site is the rate-limiting step of the combined reactions. Structural enzymology with 3S-hydroxybutanoyl-CoA shows that bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD. The flexibility of the dehydrogenase part of MFE1 further influences how substrates move between sites.
Structural diversity and stability
In simple terms: Different organisms use slightly different versions of the enzyme, but the chemistry stays the same.
The E. coli aerobic beta-oxidation trifunctional enzyme complex provides insights into stability and substrate specificity of a bacterial enoyl-CoA hydratase. Plant peroxisomal monofunctional enoyl-CoA hydratase 2 participates in the degradation of even cis-unsaturated fatty acids. Together, these studies show that the same GO:0080023 activity can be housed in monofunctional, bifunctional, or trifunctional protein architectures.
Key Genes Involved in GO:0080023 (2E)-enoyl-CoA hydratase activity
The following genes and proteins represent the main experimental models and catalytic components associated with GO:0080023.
| Gene | Major Role | Research Relevance |
|---|---|---|
| Hsd17b4 (MFE2) | Peroxisomal multifunctional enzyme 2 with enoyl-CoA hydratase activity | Bile acid synthesis and peroxisomal fatty acid oxidation |
| AtECH2 | Monofunctional peroxisomal enoyl-CoA hydratase 2 in Arabidopsis thaliana | Degradation of even cis-unsaturated fatty acids |
| MFE1 | Mitochondrial multifunctional enzyme type-1 with hydratase and dehydrogenase activities | Substrate channeling and rate-limiting dehydrogenase regeneration |
| E. coli TFE | Aerobic beta-oxidation trifunctional enzyme complex | Stability and substrate specificity of bacterial enoyl-CoA hydratase |
| M. tuberculosis TFE | Mycobacterial trifunctional enzyme with beta-oxidation activities | Substrate specificity and conformational flexibility |
| HAD | Monofunctional hydroxyacyl-CoA dehydrogenase | Comparison with bifunctional MFE1 dehydrogenase efficiency |
| ECH | Enoyl-CoA hydratase | Enantioselective hydration of non-CoA enoyl-thioesters |
| ACAD | Acyl-CoA dehydrogenase | Upstream step in beta-oxidation that generates enoyl-CoA |
| ACAA | Acetyl-CoA acyltransferase | Downstream thiolase step in beta-oxidation |
| CPT1 | Carnitine palmitoyltransferase 1 | Fatty acid entry into mitochondria for beta-oxidation |
| CPT2 | Carnitine palmitoyltransferase 2 | Fatty acid entry into mitochondria for beta-oxidation |
| SLC25A20 | Carnitine-acylcarnitine translocase | Transport of acylcarnitines for beta-oxidation |
| PPARA | Peroxisome proliferator-activated receptor alpha | Regulation of fatty acid oxidation genes |
| HADHA | Mitochondrial trifunctional protein alpha subunit | Long-chain fatty acid beta-oxidation |
| HADHB | Mitochondrial trifunctional protein beta subunit | Long-chain fatty acid beta-oxidation |
| ECI1 | Enoyl-CoA isomerase | Handling of unsaturated fatty acid intermediates |
| DECR1 | 2,4-dienoyl-CoA reductase | Reduction of dienoyl-CoA intermediates |
| ACOX1 | Peroxisomal acyl-CoA oxidase | Peroxisomal fatty acid oxidation |
How Is (2E)-enoyl-CoA hydratase activity Regulated?
The expression and activity of enzymes carrying GO:0080023 are regulated in part at the transcriptional level by nuclear receptors such as PPARA, which controls fatty acid oxidation genes in peroxisomes and mitochondria. In multifunctional enzymes, catalytic output is also regulated by substrate channeling and by the conformational flexibility of the dehydrogenase domain, which can limit the overall rate of combined reactions. The active site of enoyl-CoA hydratase can be modulated by small molecules such as 3',5'-adenosine-diphosphate, which activates the oxyanion hole and enables catalysis on non-CoA substrates.
(2E)-enoyl-CoA hydratase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| Hsd17b4 (MFE2) | Peroxisomal fatty acid oxidation and bile acid synthesis defects | Knockout cell model with lipid profiling |
| MFE1 | Mitochondrial beta-oxidation dysfunction | Point-mutation knock-in of catalytic residues |
| AtECH2 | Plant peroxisomal unsaturated fatty acid degradation | Plant knockout and overexpression lines |
| E. coli TFE | Bacterial beta-oxidation stability and substrate specificity | Bacterial knockout and complementation |
| M. tuberculosis TFE | Mycobacterial lipid metabolism | Structural and enzymatic assays |
Peroxisomal fatty acid oxidation disorders
Defects in peroxisomal multifunctional enzyme 2, which contains GO:0080023 activity, impair bile acid synthesis and the degradation of unsaturated fatty acids. Such defects can lead to accumulation of very long chain fatty acids and bile acid intermediates, contributing to peroxisomal disease phenotypes.
Mitochondrial beta-oxidation defects
Mitochondrial multifunctional enzyme type-1 (MFE1) couples enoyl-CoA hydratase activity with dehydrogenase activity, and its dysfunction can disrupt long-chain fatty acid oxidation. Structural studies suggest that inefficient dehydrogenase regeneration in MFE1 can limit the overall beta-oxidation flux.
Metabolic stress and energy homeostasis
Because GO:0080023 is required for fatty acid catabolism, its activity influences energy production during fasting and metabolic stress. Changes in the expression of fatty acid oxidation genes can alter the balance between lipid storage and oxidation.
From (2E)-enoyl-CoA hydratase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of enoyl-CoA hydratase activity impair fatty acid oxidation? | CRISPR knockout of Hsd17b4 or MFE1 in cell lines |
| Which active-site residues are required for catalysis? | Point mutation of catalytic residues followed by enzymatic assays |
| Can a disease-associated mutation be corrected? | Knock-in of wild-type sequence or base editing |
| Where is the enzyme localized in the cell? | Tagged knock-in with fluorescent protein |
| Does overexpression increase beta-oxidation flux? | Overexpression of ECH or MFE1 in mammalian cells |
| How does substrate channeling affect pathway flux? | Multifunctional enzyme mutants with disrupted channeling |
How to Study the (2E)-enoyl-CoA hydratase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enoyl-CoA hydratase assay | Catalytic hydration/dehydration rate | Kinetic characterization of wild-type and mutant enzymes |
| X-ray crystallography | Three-dimensional structure of enzyme-substrate complexes | Active-site and substrate channel analysis |
| Cryo-EM | Structure of large multifunctional complexes | Conformational flexibility studies |
| Lipidomics | Acyl-CoA and hydroxyacyl-CoA levels | Metabolic impact of enzyme loss or gain |
| CRISPR knockout screening | Gene essentiality under metabolic stress | Identification of pathway modifiers |
| Western blot | Protein expression levels | Validation of knockout or overexpression |
| Fluorescence microscopy | Subcellular localization | Peroxisomal or mitochondrial targeting |
| Site-directed mutagenesis | Residue-specific catalytic contribution | Mechanistic enzymology |
Enzymatic activity assays
Direct measurement of (2E)-enoyl-CoA hydratase activity uses spectrophotometric or chromatographic assays that follow the hydration of enoyl-CoA substrates. These assays can determine kinetic parameters and stereospecificity.
Structural biology
X-ray crystallography and cryo-EM have been used to solve structures of trifunctional and multifunctional enzymes with bound substrates, revealing active-site geometry and substrate channels. These methods help explain how mutations affect catalysis.
Lipidomics and metabolomics
Mass spectrometry-based lipidomics can quantify acyl-CoA and hydroxyacyl-CoA intermediates in cells with altered enoyl-CoA hydratase activity. This approach links enzyme function to cellular lipid homeostasis.
CRISPR-based genetic screens
Pooled CRISPR knockout screens can identify genes that modify sensitivity to fatty acid oxidation inhibitors or metabolic stress. Such screens help place GO:0080023 within broader metabolic networks.
How CRISPR Can Be Used to Study GO:0080023 (2E)-enoyl-CoA hydratase activity
Knockout
CRISPR knockout of genes encoding enoyl-CoA hydratase enzymes, such as Hsd17b4 or MFE1, can abolish GO:0080023 activity in cells. These models are used to study the consequences of blocked beta-oxidation on lipid accumulation and energy metabolism.
Point Mutation
Point mutations introduced by CRISPR base editing or homology-directed repair can target catalytic residues in the active site. Such mutants help define the contribution of individual residues to hydration and substrate specificity.
Knock-in
Knock-in of wild-type or disease-associated variants allows researchers to test whether a specific mutation alters enzyme function. Tagged knock-in with fluorescent or affinity tags enables localization and interaction studies.
Overexpression
Overexpression of enoyl-CoA hydratase genes can increase beta-oxidation flux and alter lipid profiles. These models are useful for testing whether increased activity protects against lipid stress.
How EDITGENE Supports (2E)-enoyl-CoA hydratase activity Research
Researchers studying (2E)-enoyl-CoA hydratase activity-related genes often need to determine whether a candidate gene is causally involved in fatty acid oxidation, peroxisomal metabolism, or disease phenotypes. EDITGENE provides CRISPR-based cell model services that enable precise genetic manipulation of these pathways.
Contact EDITGENE today to design your custom CRISPR model for (2E)-enoyl-CoA hydratase activity research.
Frequently Asked Questions About (2E)-enoyl-CoA hydratase activity
What is GO:0080023?
GO:0080023 is the Gene Ontology term for (2E)-enoyl-CoA hydratase activity, which catalyzes the reversible conversion of a (2E)-enoyl-CoA to a (3R)-3-hydroxyacyl-CoA and water.
What does (2E)-enoyl-CoA hydratase activity do?
It adds or removes water across the double bond of an enoyl-CoA, producing a hydroxyacyl-CoA intermediate used in fatty acid beta-oxidation.
What genes are involved in (2E)-enoyl-CoA hydratase activity?
Genes include Hsd17b4 (MFE2), MFE1, AtECH2, and bacterial trifunctional enzyme genes.
Where does (2E)-enoyl-CoA hydratase activity occur in the cell?
It occurs in peroxisomes and mitochondria, depending on the enzyme isoform.
What is the reaction catalyzed by (2E)-enoyl-CoA hydratase?
The reaction is: a (3R)-3-hydroxyacyl-CoA = a (2E)-enoyl-CoA + H2O.
How is (2E)-enoyl-CoA hydratase activity studied?
It is studied using enzymatic assays, X-ray crystallography, lipidomics, and CRISPR knockout models.
What diseases are linked to (2E)-enoyl-CoA hydratase dysfunction?
Dysfunction is linked to peroxisomal fatty acid oxidation disorders and mitochondrial beta-oxidation defects.
Can (2E)-enoyl-CoA hydratase act on non-CoA substrates?
Yes, recent work shows it can hydrate non-CoA enoyl-thioesters when the oxyanion hole is activated by 3',5'-adenosine-diphosphate.
What is the difference between MFE1 and MFE2?
MFE1 is a mitochondrial multifunctional enzyme, while MFE2 is peroxisomal and participates in bile acid synthesis.
How can CRISPR help study (2E)-enoyl-CoA hydratase activity?
CRISPR knockout, point mutation, knock-in, and overexpression models allow precise testing of gene function and catalytic residues.
Conclusion
GO:0080023, (2E)-enoyl-CoA hydratase activity, is a fundamental enzymatic function in fatty acid beta-oxidation and peroxisomal lipid metabolism. Its mechanism involves stereospecific hydration/dehydration, active-site oxyanion stabilization, and, in multifunctional enzymes, substrate channeling. Studying this activity with CRISPR models and structural methods continues to reveal how mutations and regulatory inputs affect metabolic flux and disease.
References
- 1. Qin YM et al.. 1997. Recombinant 2-enoyl-CoA hydratase derived from rat peroxisomal multifunctional enzyme 2: role of the hydratase reaction in bile acid synthesis.. Biochem J 328 ( Pt 2)(Pt 2):377-82 PMID: 9371691
- 2. Dalwani S et al.. 2026. Enantioselective Hydration of Non-CoA Enoyl-Thioesters by Enoyl-CoA Hydratase (ECH): Activation of the Active Site Oxyanion Hole with 3',5'-Adenosine-Diphosphate Enables Competent Catalysis.. JACS Au 6(4):2464-2472 PMID: 42063829
- 3. Goepfert S et al.. 2006. Identification and functional characterization of a monofunctional peroxisomal enoyl-CoA hydratase 2 that participates in the degradation of even cis-unsaturated fatty acids in Arabidopsis thaliana.. J Biol Chem 281(47):35894-903 PMID: 16982622
- 4. Dalwani S et al.. 2021. Substrate specificity and conformational flexibility properties of the Mycobacterium tuberculosis β-oxidation trifunctional enzyme.. J Struct Biol 213(3):107776 PMID: 34371166
- 5. Sridhar S et al.. 2026. Structural enzymological studies of multifunctional enzyme, type-1 (MFE1) with the 2E-decenoyl-CoA and 2E,4E-decadienoyl-CoA substrates: The regeneration of the dehydrogenase catalytic site is the rate limiting step of its combined reactions.. J Struct Biol 218(3):108346 PMID: 42435994
- 6. Sridhar S et al.. 2024. Structural enzymology studies with the substrate 3S-hydroxybutanoyl-CoA: bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD.. FEBS Open Bio 14(4):655-674 PMID: 38458818
- 7. Kasaragod P et al.. 2017. Structural enzymology comparisons of multifunctional enzyme, type-1 (MFE1): the flexibility of its dehydrogenase part.. FEBS Open Bio 7(12):1830-1842 PMID: 29226071
- 8. Sah-Teli SK et al.. 2020. Insights into the stability and substrate specificity of the E. coli aerobic β-oxidation trifunctional enzyme complex.. J Struct Biol 210(3):107494 PMID: 32171906
