GO:0120092 (2E)-butenoyl-CoA hydratase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0120092 ((2E)-butenoyl-CoA hydratase activity) catalyzes the reversible hydration of (2E)-butenoyl-CoA (crotonoyl-CoA) to 3-hydroxybutanoyl-CoA, a central step in fatty acid beta-oxidation.
• The reaction is a classic enoyl-CoA hydratase step and is also known as crotonyl-CoA hydratase activity.
• In humans, this activity is embedded in the multifunctional enzyme type-1 (MFE1), which combines enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities on a single polypeptide.
• Structural and enzymological studies of MFE1 show that the dehydrogenase catalytic site regeneration can be rate-limiting for the combined hydratase-dehydrogenase reactions.
• The flexibility of the dehydrogenase part of MFE1 is critical for substrate channeling and catalytic efficiency.
• Loss or dysfunction of this activity is linked to inherited fatty acid oxidation disorders and metabolic disease models.
Description
GO:0120092, (2E)-butenoyl-CoA hydratase activity, is a molecular function defined by the reversible hydration of (2E)-butenoyl-CoA (also called crotonoyl-CoA) to 3-hydroxybutanoyl-CoA. This reaction is a core step in the mitochondrial fatty acid beta-oxidation spiral, where it prepares the acyl-CoA intermediate for subsequent dehydrogenation. Because the same catalytic activity is also referred to as crotonyl-CoA hydratase activity, it is often studied in the context of enoyl-CoA hydratases and multifunctional enzymes. Researchers care about this term because it sits at the intersection of energy metabolism, lipid homeostasis, and inherited metabolic disease. In humans, the activity is not carried by an isolated monofunctional enzyme but is typically part of a larger multifunctional protein, such as multifunctional enzyme type-1 (MFE1), which also harbors 3-hydroxyacyl-CoA dehydrogenase activity. Understanding the catalytic mechanism, substrate specificity, and structural dynamics of this hydratase step is therefore essential for interpreting fatty acid oxidation flux and for modeling metabolic disorders. The term is also relevant to comparative enzymology, since bacterial and eukaryotic enoyl-CoA hydratases share mechanistic features but differ in assembly and regulation.
(2E)-butenoyl-CoA hydratase activity At A Glance
| GO ID | GO:0120092 |
|---|---|
| GO term | (2E)-butenoyl-CoA hydratase activity |
| Ontology | molecular_function |
| Synonym | crotonyl-CoA hydratase activity |
| Definition | Catalysis of the reaction: 3-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O; (2E)-butenoyl-CoA is also known as crotonoyl-CoA. |
| Major function | Reversible hydration of (2E)-butenoyl-CoA to 3-hydroxybutanoyl-CoA in fatty acid beta-oxidation. |
| Representative enzyme context | Multifunctional enzyme type-1 (MFE1) in humans, which combines enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. |
| Substrate | (2E)-butenoyl-CoA (crotonoyl-CoA) and 3-hydroxybutanoyl-CoA. |
| Pathway context | Mitochondrial fatty acid beta-oxidation spiral. |
| Related activity | 3-hydroxyacyl-CoA dehydrogenase activity within the same multifunctional polypeptide. |
What Is GO:0120092?
In plain terms, GO:0120092 describes an enzyme activity that adds a water molecule across the double bond of (2E)-butenoyl-CoA to produce 3-hydroxybutanoyl-CoA, and can also catalyze the reverse dehydration reaction. The official QuickGO definition states: Catalysis of the reaction: 3-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O, where (2E)-butenoyl-CoA is also known as crotonoyl-CoA. This activity is synonymous with crotonyl-CoA hydratase activity and belongs to the enoyl-CoA hydratase family. It is a molecular_function term, meaning it describes what a gene product does at the biochemical level rather than where it acts or which pathway it belongs to.
Why Is (2E)-butenoyl-CoA hydratase activity Important in Cell Biology?
GO:0120092 is important because it defines a rate-contributing step in fatty acid beta-oxidation, and its catalytic efficiency directly influences cellular energy production and lipid flux. In humans, this activity is part of multifunctional enzyme type-1 (MFE1), where the hydratase and dehydrogenase reactions are coupled; structural studies show that regeneration of the dehydrogenase catalytic site can be the rate-limiting step of the combined reactions. The flexibility of the dehydrogenase domain is also essential for substrate handling and catalysis. Consequently, perturbations in this activity can impair fatty acid oxidation and contribute to metabolic disease phenotypes, making it a relevant target for mechanistic and translational research.
• It catalyzes a central step in mitochondrial fatty acid beta-oxidation, converting (2E)-butenoyl-CoA to 3-hydroxybutanoyl-CoA.
• It is also known as crotonyl-CoA hydratase activity, linking it to classical enoyl-CoA hydratase enzymology.
• In humans, the activity is embedded in multifunctional enzyme type-1 (MFE1), which couples hydratase and dehydrogenase reactions.
• The dehydrogenase site regeneration step can be rate-limiting for the combined MFE1 reactions, making the hydratase step mechanistically coupled to overall flux.
• Domain flexibility in MFE1 is important for substrate channeling and catalytic function.
• Dysregulation of fatty acid oxidation enzymes, including this activity, is associated with inherited metabolic disorders and metabolic stress.
• The term is useful for annotating gene products in genome-scale metabolic models and for interpreting omics data.
• It provides a comparative framework for studying enoyl-CoA hydratases across species and enzyme assemblies.
• Understanding its mechanism supports structure-guided inhibitor or modulator design for metabolic targets.
• It is a key node for CRISPR-based functional studies of fatty acid oxidation genes.
Molecular Mechanism of (2E)-butenoyl-CoA hydratase activity
Substrate binding and enoyl-CoA recognition
In simple terms: The enzyme first grabs the crotonoyl-CoA molecule and positions it for reaction.
The hydratase step begins with binding of (2E)-butenoyl-CoA (crotonoyl-CoA) in the active site of an enoyl-CoA hydratase domain. Structural enzymology of multifunctional enzyme type-1 (MFE1) has shown that the enzyme accommodates 2E-decenoyl-CoA and 2E,4E-decadienoyl-CoA substrates, providing insight into how the acyl chain is positioned for hydration. The dehydrogenase part of MFE1 exhibits flexibility that supports substrate handling and channeling between active sites.
Catalytic hydration of the double bond
In simple terms: Water is added across the double bond to convert crotonoyl-CoA into 3-hydroxybutanoyl-CoA.
The defining catalytic event of GO:0120092 is the reversible hydration of (2E)-butenoyl-CoA to 3-hydroxybutanoyl-CoA. This reaction is the enoyl-CoA hydratase step of the beta-oxidation spiral and is synonymous with crotonyl-CoA hydratase activity. In MFE1, the hydratase and dehydrogenase reactions are combined on one polypeptide, and the regeneration of the dehydrogenase catalytic site can be the rate-limiting step of the combined reactions.
Coupling to 3-hydroxyacyl-CoA dehydrogenase
In simple terms: The product of the hydratase step is immediately passed to the next enzyme activity in the same protein.
In multifunctional enzyme type-1 (MFE1), the hydratase product 3-hydroxybutanoyl-CoA is channeled to the dehydrogenase active site for oxidation. Structural and enzymological studies indicate that the dehydrogenase site regeneration is rate-limiting for the combined hydratase-dehydrogenase reactions, highlighting the tight coupling between the two activities. The flexibility of the dehydrogenase part of MFE1 is important for this coupling and for overall catalytic efficiency.
Structural dynamics and substrate channeling
In simple terms: The enzyme changes shape to move intermediates between its different active sites.
Comparisons of MFE1 structures have revealed that the dehydrogenase part is flexible, which is thought to facilitate substrate channeling and the coordination of hydratase and dehydrogenase chemistry. Studies with 2E-decenoyl-CoA and 2E,4E-decadienoyl-CoA substrates have provided structural enzymological insights into how MFE1 handles different acyl-CoA species. These dynamics are central to understanding why the combined reactions can be limited by dehydrogenase site regeneration rather than by the hydratase step itself.
Reversibility and metabolic context
In simple terms: The reaction can run in either direction depending on the cell's metabolic needs.
The QuickGO definition describes the reaction as reversible: 3-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O. This reversibility allows the activity to participate in both degradative and potentially biosynthetic contexts depending on substrate availability and pathway flux. In the mitochondrial beta-oxidation spiral, the forward hydration step prepares the intermediate for subsequent dehydrogenation. The coupling with dehydrogenase activity in MFE1 ensures that the overall pathway can proceed efficiently.
Key Genes Involved in GO:0120092 (2E)-butenoyl-CoA hydratase activity
The following genes and proteins are directly or functionally associated with (2E)-butenoyl-CoA hydratase activity and its role in fatty acid oxidation, based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| HADHA | Encodes the alpha subunit of mitochondrial trifunctional protein, which contains enoyl-CoA hydratase activity including GO:0120092 | Model for fatty acid oxidation disorders and MFE1-related enzymology |
| HADHB | Encodes the beta subunit of mitochondrial trifunctional protein, contributing to the multifunctional enzyme complex | Structural and functional studies of coupled hydratase-dehydrogenase reactions |
| ECHS1 | Encodes a short-chain enoyl-CoA hydratase that can catalyze related hydratase reactions | Comparative enzymology and substrate specificity studies |
| ACAA1 | Peroxisomal thiolase involved in fatty acid oxidation pathways | Pathway context for hydratase steps in peroxisomal beta-oxidation |
| ACAA2 | Mitochondrial thiolase in beta-oxidation | Flux analysis of beta-oxidation spiral |
| ACADVL | Very long-chain acyl-CoA dehydrogenase, upstream of hydratase steps | Model for fatty acid oxidation disorders |
| ACADM | Medium-chain acyl-CoA dehydrogenase, upstream of hydratase steps | Metabolic disease modeling |
| CPT1A | Rate-limiting enzyme for mitochondrial fatty acid import | Upstream regulation of beta-oxidation flux |
| CPT2 | Inner mitochondrial membrane carnitine palmitoyltransferase | Fatty acid oxidation disorder models |
| SLC25A20 | Carnitine-acylcarnitine translocase | Transport step affecting beta-oxidation substrate supply |
| HADH | Hydroxyacyl-CoA dehydrogenase, downstream of hydratase step | Coupling of hydratase and dehydrogenase activities |
| ETFA | Electron transfer flavoprotein alpha subunit | Electron transfer from acyl-CoA dehydrogenases |
| ETFB | Electron transfer flavoprotein beta subunit | Electron transfer chain in beta-oxidation |
| ETFDH | Electron transfer flavoprotein dehydrogenase | Mitochondrial electron transfer for beta-oxidation |
| PPARA | Nuclear receptor regulating fatty acid oxidation gene expression | Transcriptional regulation of beta-oxidation genes |
| PPARGC1A | Coactivator regulating mitochondrial biogenesis and fatty acid oxidation | Regulation of oxidative metabolism |
| NRF1 | Transcription factor controlling mitochondrial biogenesis | Regulation of fatty acid oxidation capacity |
How Is (2E)-butenoyl-CoA hydratase activity Regulated?
The expression and activity of enzymes containing (2E)-butenoyl-CoA hydratase activity are regulated at multiple levels. Transcriptional control of fatty acid oxidation genes is mediated by nuclear receptors such as PPARA and coactivators like PPARGC1A, which respond to metabolic demand. At the protein level, the catalytic efficiency of multifunctional enzyme type-1 (MFE1) is influenced by the structural coupling between its hydratase and dehydrogenase domains, with dehydrogenase site regeneration being rate-limiting for the combined reactions. The flexibility of the dehydrogenase part of MFE1 further modulates substrate channeling and overall activity. These regulatory features ensure that the hydratase step is coordinated with upstream fatty acid import and downstream electron transfer.
(2E)-butenoyl-CoA hydratase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| HADHA | Fatty acid oxidation disorder, mitochondrial trifunctional protein deficiency | Knockout or point-mutation cell models in hepatocytes or fibroblasts |
| HADHB | Mitochondrial trifunctional protein deficiency | Knockout cell lines and metabolic flux assays |
| ECHS1 | Short-chain enoyl-CoA hydratase deficiency, metabolic disease | Knockout and overexpression models in patient-derived cells |
| ACADVL | Very long-chain acyl-CoA dehydrogenase deficiency | Point-mutation knock-in models for enzyme kinetics |
| PPARA | Metabolic regulation and fatty acid oxidation disorders | Knockout and overexpression models for transcriptional studies |
Inherited fatty acid oxidation disorders
Defects in mitochondrial fatty acid beta-oxidation, including enzymes that harbor (2E)-butenoyl-CoA hydratase activity, can lead to inherited metabolic disorders characterized by impaired energy production during fasting or illness. The multifunctional enzyme type-1 (MFE1) couples hydratase and dehydrogenase activities, and disruption of this coupling can compromise the entire beta-oxidation spiral. Structural studies showing that dehydrogenase site regeneration is rate-limiting provide a mechanistic basis for understanding how partial defects may manifest clinically.
Metabolic stress and energy homeostasis
Because (2E)-butenoyl-CoA hydratase activity is a core step in fatty acid oxidation, its dysfunction can contribute to metabolic stress and altered energy homeostasis. The flexibility of the dehydrogenase domain in MFE1 is important for adapting to different substrate loads, and perturbations may affect flux through the pathway. Research models with altered hydratase activity can help dissect the contribution of this step to metabolic phenotypes.
Cancer metabolism and lipid dependency
Altered fatty acid oxidation is increasingly recognized as a feature of cancer metabolism, and enzymes involved in beta-oxidation, including hydratase steps, may influence tumor cell survival under metabolic stress. The coupling of hydratase and dehydrogenase activities in MFE1 suggests that targeting this multifunctional enzyme could affect multiple steps of the pathway. However, direct evidence linking GO:0120092 specifically to cancer requires further study using appropriate models.
From (2E)-butenoyl-CoA hydratase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does loss of hydratase activity impair beta-oxidation flux? | CRISPR knockout of HADHA or HADHB in hepatocyte or fibroblast cell lines |
| How do point mutations affect catalytic efficiency? | Point-mutation knock-in of catalytic residues in MFE1 domains |
| Can tagged hydratase domains be used for interaction studies? | Knock-in of epitope-tagged HADHA or HADHB |
| Does overexpression alter lipid metabolism? | Overexpression of HADHA or HADHB in metabolic cell models |
| How does dehydrogenase domain flexibility affect coupled reactions? | Knock-in of flexible-loop variants and structural enzymology |
| What is the rate-limiting step in combined reactions? | Point mutations at dehydrogenase site and kinetic assays |
How to Study the (2E)-butenoyl-CoA hydratase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Spectrophotometric hydratase assay | Rate of crotonoyl-CoA hydration | Kinetic characterization of wild-type and mutant enzymes |
| Coupled dehydrogenase assay | Combined hydratase-dehydrogenase flux | Determining rate-limiting steps |
| X-ray crystallography | Three-dimensional structure of enzyme-substrate complexes | Structural enzymology of MFE1 |
| Site-directed mutagenesis | Effect of specific residues on catalysis | Mechanistic studies of active site |
| Metabolic flux analysis | Pathway flux through beta-oxidation | Assessing impact of gene edits |
| Transcriptomics | Expression of fatty acid oxidation genes | Regulatory studies under metabolic stress |
| Proteomics | Protein abundance and modifications | Validation of knockout or overexpression models |
| Bioinformatics | Sequence conservation and variant prediction | Target prioritization for CRISPR studies |
Enzymatic activity assays
Direct measurement of (2E)-butenoyl-CoA hydratase activity can be performed using spectrophotometric assays that monitor the hydration of crotonoyl-CoA to 3-hydroxybutanoyl-CoA. These assays are essential for determining kinetic parameters and for comparing wild-type and mutant enzymes. Coupled assays that include the dehydrogenase step can reveal whether dehydrogenase site regeneration is rate-limiting.
Structural enzymology
X-ray crystallography and structural enzymology of multifunctional enzyme type-1 (MFE1) with substrates such as 2E-decenoyl-CoA and 2E,4E-decadienoyl-CoA provide atomic-level insights into substrate binding and catalysis. Comparisons of different MFE1 structures reveal the flexibility of the dehydrogenase part, which is important for substrate channeling. These methods help explain how mutations affect catalytic function.
Metabolic flux analysis
Metabolic flux analysis using labeled substrates or mass spectrometry can quantify the contribution of the hydratase step to overall fatty acid oxidation. Such approaches are useful for assessing the impact of CRISPR-generated mutations on pathway flux. Combining flux data with enzyme kinetics provides a systems-level view of beta-oxidation.
Omics and bioinformatics
Transcriptomics and proteomics can reveal changes in expression of genes encoding hydratase-containing enzymes under different metabolic conditions. Bioinformatics analysis of enzyme families can identify conserved motifs and predict functional consequences of variants. Integrating omics data with structural information helps prioritize targets for experimental validation.
How CRISPR Can Be Used to Study GO:0120092 (2E)-butenoyl-CoA hydratase activity
Knockout
CRISPR knockout of genes encoding enzymes with (2E)-butenoyl-CoA hydratase activity, such as HADHA or HADHB, can be used to assess the contribution of this activity to fatty acid oxidation flux. Knockout cell models allow researchers to measure compensatory changes in other beta-oxidation enzymes and to test metabolic dependencies. These models are particularly useful for studying inherited metabolic disorders linked to impaired fatty acid oxidation.
Point Mutation
Point-mutation knock-in using CRISPR can introduce specific amino acid substitutions in the active site of the hydratase or dehydrogenase domains to dissect catalytic mechanisms. Such models help determine whether a particular residue is essential for hydration or for coupling to the dehydrogenase step. They are also valuable for validating structural predictions from crystallography.
Knock-in
Knock-in of epitope tags or fluorescent reporters into endogenous HADHA or HADHB loci enables real-time tracking of protein localization and interactions. Tagged knock-in models can be used to study substrate channeling between hydratase and dehydrogenase active sites. These models are also useful for proteomic pull-down experiments to identify interacting partners.
Overexpression
Overexpression of genes containing (2E)-butenoyl-CoA hydratase activity can be used to test whether increased enzyme levels alter lipid metabolism or metabolic flux. Overexpression models are helpful for studying dose-dependent effects and for producing recombinant enzyme for biochemical assays. They can also reveal dominant-negative effects of mutant variants.
How EDITGENE Supports (2E)-butenoyl-CoA hydratase activity Research
Researchers studying (2E)-butenoyl-CoA hydratase activity-related genes often need to determine whether a candidate gene is causally involved in fatty acid oxidation, metabolic disease, or cellular stress responses. Rigorous causal inference requires well-controlled genetic models that can isolate the contribution of specific enzymatic activities from compensatory pathways. EDITGENE provides a suite of CRISPR-based services designed to support such studies, from knockout to precise point mutations and knock-in reporters.
Contact EDITGENE today to design your custom CRISPR model for (2E)-butenoyl-CoA hydratase activity research.
Frequently Asked Questions About (2E)-butenoyl-CoA hydratase activity
What is GO:0120092?
GO:0120092 is the Gene Ontology term for (2E)-butenoyl-CoA hydratase activity, which catalyzes the reversible hydration of (2E)-butenoyl-CoA to 3-hydroxybutanoyl-CoA.
What is (2E)-butenoyl-CoA hydratase activity?
It is an enzyme activity that adds water to crotonoyl-CoA to form 3-hydroxybutanoyl-CoA, a step in fatty acid beta-oxidation.
What genes are involved in (2E)-butenoyl-CoA hydratase activity?
Genes such as HADHA and HADHB encode multifunctional enzymes that contain this activity in humans.
What is another name for (2E)-butenoyl-CoA hydratase activity?
It is also known as crotonyl-CoA hydratase activity.
Which pathway uses (2E)-butenoyl-CoA hydratase activity?
It is a core step in the mitochondrial fatty acid beta-oxidation spiral.
How is (2E)-butenoyl-CoA hydratase activity studied?
It is studied using enzymatic assays, structural enzymology, and metabolic flux analysis.
What diseases are linked to defects in this activity?
Defects in fatty acid oxidation enzymes, including those with this activity, are linked to inherited metabolic disorders.
Is (2E)-butenoyl-CoA hydratase activity reversible?
Yes, the reaction is reversible: 3-hydroxybutanoyl-CoA = (2E)-butenoyl-CoA + H2O.
What is the role of MFE1 in this activity?
Multifunctional enzyme type-1 (MFE1) combines enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities on one polypeptide.
How can CRISPR help study (2E)-butenoyl-CoA hydratase activity?
CRISPR knockout, point mutation, knock-in, and overexpression models can be used to dissect the function of genes encoding this activity.
Conclusion
GO:0120092, (2E)-butenoyl-CoA hydratase activity, is a fundamental enzymatic step in fatty acid beta-oxidation, catalyzing the reversible hydration of crotonoyl-CoA to 3-hydroxybutanoyl-CoA. In humans, this activity is embedded in multifunctional enzyme type-1 (MFE1), where it is tightly coupled to dehydrogenase activity, with dehydrogenase site regeneration being rate-limiting for the combined reactions. Structural studies highlight the importance of dehydrogenase domain flexibility for substrate channeling and catalysis. Understanding this activity is essential for interpreting metabolic flux, modeling fatty acid oxidation disorders, and designing targeted experiments. CRISPR-based models, including knockout, point mutation, knock-in, and overexpression, provide powerful tools to investigate the causal roles of genes harboring this activity in health and disease.
References
- 1. 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
- 2. 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
