GO:0004300 enoyl-CoA hydratase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004300 enoyl-CoA hydratase activity catalyzes the reversible hydration of enoyl-CoA to 3-hydroxyacyl-CoA, a central step in fatty acid beta-oxidation.
• The reaction proceeds through a concerted mechanism involving a conserved glutamate as a general acid/base and a water molecule.
• ECHS1 (short-chain enoyl-CoA hydratase) is the best-characterized human enzyme carrying this activity, and its deficiency causes a severe mitochondrial disease.
• ECHS1 also regulates mTOR signaling and apoptosis by sensing nutrients, linking enoyl-CoA hydratase activity to cell growth control.
• Enoyl-CoA hydratase activity is inhibited by acetylation of lysine residues, connecting cellular metabolism to post-translational modifications.
• Studying GO:0004300 requires integrating structural biology, enzymology, and CRISPR-based models to dissect its roles in health and disease.
Description
Enoyl-CoA hydratase activity (GO:0004300) is a fundamental enzymatic function in fatty acid metabolism, catalyzing the reversible hydration of enoyl-CoA to 3-hydroxyacyl-CoA. This reaction is a key step in the mitochondrial beta-oxidation spiral, where it facilitates the breakdown of fatty acids to generate acetyl-CoA and reducing equivalents. The enzyme is highly conserved across species, from bacteria to humans, underscoring its essential role in energy homeostasis. In humans, short-chain enoyl-CoA hydratase (ECHS1) is the primary enzyme exhibiting this activity, and mutations in ECHS1 lead to a devastating neurometabolic disorder. Beyond its canonical role, enoyl-CoA hydratase activity has been implicated in the regulation of mTOR signaling and apoptosis, suggesting a broader function in cellular nutrient sensing. Understanding the molecular details of GO:0004300 is therefore critical for both basic biochemistry and clinical research.
enoyl-CoA hydratase activity At A Glance
| GO ID | GO:0004300 |
|---|---|
| GO term | enoyl-CoA hydratase activity |
| Ontology | molecular_function |
| Synonym | 2-enoyl-CoA hydratase activity; 3-hydroxyacyl-CoA dehydratase; short-chain enoyl-CoA hydratase activity; enoyl hydrase activity |
| Major function | Catalyzes the reversible hydration of enoyl-CoA to 3-hydroxyacyl-CoA, a step in fatty acid beta-oxidation |
| Reaction direction | Usually occurs in reverse, reducing the double bond of enoyl-CoA at position 2 or 3 |
| Substrates | Enoyl-CoA derivatives, including (3E)-enoyl-CoA and (2E)-enoyl-CoA |
| Products | 3-hydroxy-fatty acyl-CoA, including (3S)-3-hydroxyacyl-CoA |
| Cofactors | None required; uses a water molecule and a conserved glutamate as general acid/base |
What Is GO:0004300?
GO:0004300 enoyl-CoA hydratase activity is defined as the catalysis of the reversible reaction: a 3-hydroxy-fatty acyl-CoA = an enoyl-CoA + H2O. In the physiological direction, it adds water across the double bond of enoyl-CoA to form 3-hydroxyacyl-CoA, but the reaction can also proceed in reverse to dehydrate 3-hydroxyacyl-CoA, generating enoyl-CoA. The reaction typically occurs at the second or third carbon of the acyl chain, and specific substrates include 4-saturated-(3S)-3-hydroxyacyl-CoA and (3S)-3-hydroxyacyl-CoA, yielding (3E)-enoyl-CoA and (2E)-enoyl-CoA, respectively. This activity is synonymous with short-chain enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydratase, and enoyl hydrase, among others.
Why Is enoyl-CoA hydratase activity Important in Cell Biology?
Enoyl-CoA hydratase activity is indispensable for fatty acid beta-oxidation, a central energy-producing pathway in mitochondria. Defects in this activity cause ECHS1 deficiency, a severe autosomal recessive disorder characterized by developmental delay, metabolic acidosis, and early death. The enzyme also plays a role in nutrient sensing and apoptosis regulation, linking metabolism to cell fate decisions. Moreover, enoyl-CoA hydratase activity is a target for metabolic engineering, such as the biosynthesis of vanillin. Thus, understanding GO:0004300 has broad implications for human health, biotechnology, and fundamental cell biology.
• Essential for mitochondrial fatty acid beta-oxidation and energy production.
• Mutations in ECHS1 cause a severe neurometabolic disorder with high mortality.
• Regulates mTOR signaling and apoptosis in response to nutrient availability.
• Acetylation of lysine residues inhibits enoyl-CoA hydratase activity, linking metabolism to post-translational modifications.
• Serves as a biocatalyst for the production of vanillin and other chemicals.
• Highly conserved across evolution, from Thermus thermophilus to humans.
• Involved in the mitochondrial trifunctional protein complex, which is activated by spermidine to improve antitumor immunity.
• Provides a model system for studying enzyme mechanism and inhibition.
• Potential target for treating metabolic disorders and cancer.
• Enables structural and biochemical studies of substrate recognition.
What Happens During enoyl-CoA hydratase activity?
Substrate Binding and Orientation
In simple terms: The enzyme grabs the enoyl-CoA molecule and positions it perfectly for a chemical reaction.
The first step in enoyl-CoA hydratase activity is the binding of the enoyl-CoA substrate to the active site. Structural studies of enoyl-CoA hydratase from Thermus thermophilus HB8 reveal a conserved fold that accommodates the acyl chain and CoA moiety. In human ECHS1, substrate recognition involves specific residues that orient the double bond for hydration. The enzyme typically functions as a homodimer or as part of a multifunctional complex, with each subunit contributing to substrate binding.
Catalytic Hydration/Dehydration
In simple terms: A water molecule is added across the double bond, or removed, to convert between two forms of the molecule.
The catalytic mechanism involves a conserved glutamate residue acting as a general acid/base. In the hydratase direction, water is added to the double bond of enoyl-CoA, forming 3-hydroxyacyl-CoA. In the reverse direction, a proton is abstracted from the 3-hydroxy group, and a water molecule is eliminated to regenerate the enoyl-CoA. The reaction is stereospecific, producing (3S)-3-hydroxyacyl-CoA from (2E)-enoyl-CoA. This step is reversible and is driven by substrate availability and the overall metabolic state.
Product Release and Channeling
In simple terms: The newly formed product is released or passed directly to the next enzyme in the pathway.
After catalysis, the 3-hydroxyacyl-CoA product is released from the active site. In mitochondria, enoyl-CoA hydratase is part of the trifunctional protein (TFP) complex, which also contains long-chain 3-hydroxyacyl-CoA dehydrogenase and long-chain 3-ketoacyl-CoA thiolase. This organization allows substrate channeling, where the product of the hydratase reaction is directly transferred to the next enzyme, enhancing pathway efficiency. In ECHS1, product release is facilitated by conformational changes in the active site.
Regulation by Acetylation and Nutrients
In simple terms: The enzyme's activity can be turned on or off by chemical tags or by the availability of nutrients.
Enoyl-CoA hydratase activity is regulated by post-translational modifications. Acetylation of lysine residues on metabolic enzymes, including enoyl-CoA hydratase, can inhibit their activity, as shown in a global study of protein lysine acetylation. Additionally, ECHS1 acts as a nutrient sensor, regulating mTOR signaling and apoptosis in response to changes in cellular energy status. This regulation links enoyl-CoA hydratase activity to cell growth and survival decisions.
Key Genes Involved in GO:0004300 enoyl-CoA hydratase activity
The following genes and proteins are directly associated with enoyl-CoA hydratase activity (GO:0004300) or its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ECHS1 | Short-chain enoyl-CoA hydratase; primary human enzyme with GO:0004300 activity | Mutations cause ECHS1 deficiency; regulates mTOR and apoptosis |
| HADHA | Long-chain enoyl-CoA hydratase subunit of mitochondrial trifunctional protein | Defects cause long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; activated by spermidine |
| HADHB | Long-chain 3-ketoacyl-CoA thiolase subunit of trifunctional protein | Forms complex with HADHA; involved in fatty acid oxidation |
| ACAA2 | Mitochondrial 3-ketoacyl-CoA thiolase; interacts with enoyl-CoA hydratase | Part of beta-oxidation pathway; potential target for metabolic studies |
| ACADM | Medium-chain acyl-CoA dehydrogenase; upstream of enoyl-CoA hydratase | Deficiency causes MCAD deficiency; model for pathway analysis |
| ACADS | Short-chain acyl-CoA dehydrogenase; upstream of enoyl-CoA hydratase | Deficiency linked to metabolic disorders |
| ACADVL | Very long-chain acyl-CoA dehydrogenase; upstream of enoyl-CoA hydratase | Deficiency causes VLCAD deficiency; part of beta-oxidation |
| CPT1A | Carnitine palmitoyltransferase 1A; regulates entry of fatty acids into mitochondria | Rate-limiting for beta-oxidation; target for metabolic regulation |
| CPT2 | Carnitine palmitoyltransferase 2; inner mitochondrial membrane | Deficiency causes CPT II deficiency; affects beta-oxidation flux |
| SLC25A20 | Carnitine-acylcarnitine translocase; transports fatty acyl-carnitines | Deficiency impairs beta-oxidation; model for transport studies |
| PPARA | Peroxisome proliferator-activated receptor alpha; transcription factor regulating beta-oxidation genes | Controls expression of ECHS1 and other beta-oxidation enzymes |
| PPARGC1A | PGC-1alpha; coactivator regulating mitochondrial biogenesis and fatty acid oxidation | Modulates beta-oxidation capacity; target for metabolic engineering |
| SIRT1 | NAD+-dependent deacetylase; regulates acetylation of metabolic enzymes | Deacetylates and activates enoyl-CoA hydratase |
| SIRT3 | Mitochondrial deacetylase; regulates acetylation of beta-oxidation enzymes | Modulates enoyl-CoA hydratase activity via deacetylation |
| EP300 | Histone acetyltransferase; acetylates metabolic enzymes | Inhibits enoyl-CoA hydratase activity by acetylation |
| CREBBP | Histone acetyltransferase; acetylates metabolic enzymes | Regulates enoyl-CoA hydratase via acetylation |
| MTOR | Serine/threonine kinase; regulated by ECHS1 nutrient sensing | ECHS1 regulates mTOR signaling |
| BCL2 | Anti-apoptotic protein; modulated by ECHS1 | ECHS1 regulates apoptosis via BCL2 |
How Is enoyl-CoA hydratase activity Regulated?
Enoyl-CoA hydratase activity is regulated at multiple levels. Post-translational acetylation of lysine residues inhibits enzyme activity, and this modification is controlled by acetyltransferases (EP300, CREBBP) and deacetylases (SIRT1, SIRT3). Nutrient availability influences ECHS1 function, which in turn regulates mTOR signaling and apoptosis. Additionally, the mitochondrial trifunctional protein, which contains enoyl-CoA hydratase activity, is activated by spermidine, linking polyamine metabolism to fatty acid oxidation. Transcriptional regulation by PPARA and PPARGC1A also controls the expression of beta-oxidation genes, including ECHS1.
enoyl-CoA hydratase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| ECHS1 | ECHS1 deficiency (neurometabolic disorder) | ECHS1 knockout or point-mutation cell lines; patient-derived fibroblasts |
| HADHA | Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency | HADHA knockout HepG2 cells; mitochondrial function assays |
| HADHB | Trifunctional protein deficiency | HADHB knockout cell models; fatty acid oxidation flux |
| ECHS1 | Cancer (mTOR signaling, apoptosis) | ECHS1 overexpression or knockout in cancer cell lines; mTOR reporter assays |
| SIRT3 | Metabolic regulation via deacetylation | SIRT3 knockout or overexpression; acetylation profiling |
ECHS1 Deficiency: A Severe Neurometabolic Disorder
Biallelic mutations in ECHS1 cause short-chain enoyl-CoA hydratase deficiency, an autosomal recessive disorder characterized by developmental delay, progressive neurodegeneration, metabolic acidosis, and often early death. The disease is particularly prevalent in the Pacific population, where a founder mutation has been identified. Biochemical studies show that mutant ECHS1 proteins have reduced hydratase activity, leading to impaired fatty acid oxidation and accumulation of toxic metabolites. This condition highlights the critical role of GO:0004300 in human health.
Enoyl-CoA Hydratase Activity in Cancer and Nutrient Sensing
ECHS1 regulates mTOR signaling and apoptosis by sensing nutrients, suggesting a role in cancer cell metabolism. Loss of ECHS1 can lead to uncontrolled mTOR activation and resistance to apoptosis, promoting tumor growth. Additionally, the mitochondrial trifunctional protein, which includes enoyl-CoA hydratase activity, is activated by spermidine and improves antitumor immunity in mice. These findings indicate that modulating enoyl-CoA hydratase activity could be a therapeutic strategy in cancer.
Metabolic Engineering and Biocatalysis
Enoyl-CoA hydratase activity is exploited in biotechnology for the biosynthesis of vanillin. Rational design of enoyl-CoA hydratase/lyase enzymes has enabled efficient conversion of ferulic acid to vanillin. This application demonstrates the industrial potential of GO:0004300 and the importance of understanding its substrate specificity and mechanism.
From enoyl-CoA hydratase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of ECHS1 loss on fatty acid oxidation? | ECHS1 knockout cell line (e.g., HEK293T, HeLa) |
| How do patient mutations affect enoyl-CoA hydratase activity? | Point-mutation knock-in of ECHS1 variants (e.g., c.476A>G) |
| Can ECHS1 be tagged for localization studies? | Knock-in of FLAG- or GFP-ECHS1 at endogenous locus |
| Does ECHS1 overexpression alter mTOR signaling? | ECHS1 overexpression in cancer cell lines |
| What is the role of acetylation in regulating enoyl-CoA hydratase? | Knock-in of acetylation-deficient or -mimetic ECHS1 mutants |
| Can enoyl-CoA hydratase activity be redirected for vanillin production? | Overexpression of engineered enoyl-CoA hydratase/lyase in E. coli |
How to Study the enoyl-CoA hydratase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric assay | Enoyl-CoA hydratase activity (decrease in A263) | Kinetic characterization of wild-type and mutant enzymes |
| X-ray crystallography | Three-dimensional structure of enzyme-substrate complex | Mechanistic studies and inhibitor design |
| 13C metabolic flux analysis | Flux through beta-oxidation | Quantifying pathway activity in cells |
| CRISPR knockout screening | Genes affecting cell fitness under ECHS1 perturbation | Identifying synthetic lethal partners |
| Western blot | Protein expression and acetylation status | Validating knockout or overexpression |
| Immunofluorescence | Subcellular localization of ECHS1 | Confirming mitochondrial targeting |
| RNA-seq | Transcriptional changes upon ECHS1 modulation | Pathway analysis and biomarker discovery |
| Co-immunoprecipitation | Protein-protein interactions of ECHS1 | Identifying binding partners in beta-oxidation complex |
Enzymatic Activity Assays
Direct measurement of enoyl-CoA hydratase activity is performed using spectrophotometric assays that monitor the decrease in absorbance at 263 nm as enoyl-CoA is hydrated to 3-hydroxyacyl-CoA. These assays can be adapted for high-throughput screening of inhibitors or mutants. For ECHS1, recombinant protein can be purified and assayed with various enoyl-CoA substrates to determine kinetic parameters.
Structural Biology
X-ray crystallography and cryo-EM have been used to determine the structures of enoyl-CoA hydratases from Thermus thermophilus and humans. These structures reveal the active site architecture, substrate binding mode, and conformational changes during catalysis. Structural studies are essential for understanding mechanism and for rational drug design.
Metabolic Flux Analysis
Isotope tracing with 13C-labeled fatty acids coupled to mass spectrometry can measure flux through beta-oxidation in cells with altered enoyl-CoA hydratase activity. This approach quantifies the contribution of GO:0004300 to overall mitochondrial metabolism and can reveal compensatory pathways.
CRISPR Screening and Functional Genomics
Genome-wide CRISPR knockout screens can identify genes that modulate enoyl-CoA hydratase activity or its downstream effects. For example, screens for resistance to ECHS1 deficiency-induced stress can uncover synthetic lethal interactions. These methods are powerful for discovering new regulators and therapeutic targets.
How CRISPR Can Be Used to Study GO:0004300 enoyl-CoA hydratase activity
Knockout
CRISPR-Cas9 knockout of ECHS1 or other genes encoding enoyl-CoA hydratase activity enables the study of loss-of-function phenotypes. ECHS1 knockout cell lines exhibit impaired fatty acid oxidation, accumulation of 3-hydroxyacylcarnitines, and altered mTOR signaling. These models are valuable for dissecting the metabolic and signaling roles of GO:0004300.
Point Mutation
Introducing patient-specific point mutations into the endogenous ECHS1 locus using CRISPR base editing or homology-directed repair allows precise modeling of ECHS1 deficiency. For example, the c.476A>G (p.Asn159Ser) mutation found in the Pacific population can be knocked in to study its effect on enzyme activity and cellular metabolism. Point-mutation models are essential for understanding genotype-phenotype correlations.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins (e.g., GFP) at the ECHS1 locus enables real-time tracking of protein localization and interaction. Tagged knock-in cell lines can be used for live-cell imaging, immunoprecipitation, and proteomics to uncover new functions of enoyl-CoA hydratase activity.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of ECHS1 can be used to study gain-of-function effects. Overexpression of ECHS1 in cancer cell lines modulates mTOR signaling and apoptosis, providing insights into its role in tumor metabolism. Overexpression is also useful for producing recombinant enzyme for structural and biochemical studies.
How EDITGENE Supports enoyl-CoA hydratase activity Research
Researchers studying enoyl-CoA hydratase activity-related genes often need to determine whether a candidate gene is causally involved in metabolic regulation, disease pathogenesis, or drug response. EDITGENE provides a comprehensive suite of CRISPR-based services to create precisely engineered cell models, enabling rigorous functional validation of GO:0004300 and its associated pathways.
Contact EDITGENE today to design your custom CRISPR model for enoyl-CoA hydratase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| HADHA Knockout HEK293 Cell Line | EDJ-KQ2238 | Human | 3030 | Details Get a Quote |
| AUH Knockout HEK293 Cell Line | EDJ-KQ2744 | Human | 549 | Details Get a Quote |
| ECHS1 Knockout HEK293 Cell Line | EDJ-KQ4495 | Human | 1892 | Details Get a Quote |
| EHHADH Knockout HEK293 Cell Line | EDJ-KQ4507 | Human | 1962 | Details Get a Quote |
| HADHB Knockout HEK293 Cell Line | EDJ-KQ4838 | Human | 3032 | Details Get a Quote |
| HSD17B4 Knockout HEK293 Cell Line | EDJ-KQ4941 | Human | 3295 | Details Get a Quote |
| ECHDC3 Knockout HEK293 Cell Line | EDJ-KQ12472 | Human | 79746 | Details Get a Quote |
| ECHDC2 Knockout HEK293 Cell Line | EDJ-KQ13248 | Human | 55268 | Details Get a Quote |
| AUH Knockout A-549 Cell Line | EDJ-KQ25013 | Human | 549 | Details Get a Quote |
| AUH Knockout HCT 116 Cell Line | EDJ-KQ25015 | Human | 549 | Details Get a Quote |
| AUH Knockout HeLa Cell Line | EDJ-KQ25016 | Human | 549 | Details Get a Quote |
| ECHS1 Knockout A-549 Cell Line | EDJ-KQ27075 | Human | 1892 | Details Get a Quote |
| ECHS1 Knockout HeLa Cell Line | EDJ-KQ27077 | Human | 1892 | Details Get a Quote |
| EHHADH Knockout A-549 Cell Line | EDJ-KQ27111 | Human | 1962 | Details Get a Quote |
| EHHADH Knockout HCT 116 Cell Line | EDJ-KQ27112 | Human | 1962 | Details Get a Quote |
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Frequently Asked Questions About enoyl-CoA hydratase activity
What is enoyl-CoA hydratase activity?
Enoyl-CoA hydratase activity (GO:0004300) is a molecular function that catalyzes the reversible hydration of enoyl-CoA to 3-hydroxyacyl-CoA, a key step in fatty acid beta-oxidation.
What genes are involved in enoyl-CoA hydratase activity?
The primary human gene is ECHS1, but HADHA and HADHB also encode subunits with this activity in the mitochondrial trifunctional protein.
What diseases are associated with enoyl-CoA hydratase deficiency?
Mutations in ECHS1 cause a severe neurometabolic disorder with developmental delay and metabolic acidosis. Defects in HADHA/HADHB cause long-chain fatty acid oxidation disorders.
How is enoyl-CoA hydratase activity regulated?
It is regulated by acetylation of lysine residues, which inhibits activity, and by nutrient sensing through mTOR signaling.
What is the reaction mechanism of enoyl-CoA hydratase?
The enzyme uses a conserved glutamate as a general acid/base to add or remove water across the double bond of enoyl-CoA, forming or breaking the 3-hydroxyacyl-CoA.
Can enoyl-CoA hydratase be used in biotechnology?
Yes, engineered enoyl-CoA hydratase/lyase enzymes are used for the biosynthesis of vanillin from ferulic acid.
What model systems are used to study enoyl-CoA hydratase activity?
Common models include ECHS1 knockout cell lines, patient-derived fibroblasts, and recombinant protein for structural and kinetic studies.
How does ECHS1 regulate mTOR signaling?
ECHS1 senses nutrient availability and modulates mTOR signaling and apoptosis, linking fatty acid oxidation to cell growth control.
What is the role of enoyl-CoA hydratase in cancer?
ECHS1 regulates apoptosis and mTOR, and its loss can promote tumor growth; the trifunctional protein is activated by spermidine to improve antitumor immunity.
How can CRISPR be used to study enoyl-CoA hydratase activity?
CRISPR knockout, point mutation knock-in, and overexpression models allow precise dissection of gene function in metabolic pathways and disease.
Conclusion
Enoyl-CoA hydratase activity (GO:0004300) is a cornerstone of fatty acid metabolism with far-reaching implications for human health and disease. From its conserved catalytic mechanism to its roles in ECHS1 deficiency, cancer, and nutrient sensing, this enzymatic activity continues to be a rich area of research. Advances in CRISPR-based models and structural biology are poised to uncover new therapeutic opportunities targeting this pathway.
References
- 1. Padavattan S et al.. 2021. Crystal structure of enoyl-CoA hydratase from Thermus thermophilus HB8.. Acta Crystallogr F Struct Biol Commun 77(Pt 5):148-155 PMID: 33949975
- 2. Zhao S et al.. 2010. Regulation of cellular metabolism by protein lysine acetylation.. Science 327(5968):1000-4 PMID: 20167786
- 3. Agnihotri G et al.. 2003. Enoyl-CoA hydratase. reaction, mechanism, and inhibition.. Bioorg Med Chem 11(1):9-20 PMID: 12467702
- 4. Su G et al.. 2025. Structural and biochemical mechanism of short-chain enoyl-CoA hydratase (ECHS1) substrate recognition.. Commun Biol 8(1):619 PMID: 40240482
- 5. Ye Q et al.. 2023. Biosynthesis of Vanillin by Rational Design of Enoyl-CoA Hydratase/Lyase.. Int J Mol Sci 24(17) PMID: 37686435
- 6. Bernhardt I et al.. 2024. Further delineation of short-chain enoyl-CoA hydratase deficiency in the Pacific population.. Mol Genet Metab 142(3):108508 PMID: 38820906
- 7. Al-Habsi M et al.. 2022. Spermidine activates mitochondrial trifunctional protein and improves antitumor immunity in mice.. Science 378(6618):eabj3510 PMID: 36302005
- 8. Zhang YK et al.. 2017. Enoyl-CoA hydratase-1 regulates mTOR signaling and apoptosis by sensing nutrients.. Nat Commun 8(1):464 PMID: 28878358
