GO:0047977 hepoxilin-epoxide hydrolase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047977 (hepoxilin-epoxide hydrolase activity) catalyzes the hydrolysis of hepoxilin A3 to its corresponding trihydroxy metabolite, a reaction first purified from rat liver.
• Mammalian soluble epoxide hydrolase (sEH, gene EPHX2) is identical to liver hepoxilin hydrolase, linking this activity to a well-characterized xenobiotic and lipid-metabolizing enzyme.
• Hepoxilin A3 is formed in the rat aorta and pineal gland and is metabolized either by epoxide hydrolase or by glutathione conjugation to HxA3-C.
• The enzyme belongs to the epoxide hydrolase family, which plays broad roles in lipid mediator metabolism and detoxification.
• Dysregulation of hepoxilin/epoxide hydrolase activity has been implicated in inflammation, vascular biology, and central nervous system signaling.
• CRISPR-based knockout, point-mutation, and knock-in models of EPHX2 and related genes enable precise dissection of hepoxilin-epoxide hydrolase function in health and disease.
Description
Hepoxilin-epoxide hydrolase activity (GO:0047977) is a molecular function defined as the catalysis of the reaction (5Z,9E,14Z)-(8x,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate + H2O = (5Z,9E,14Z)-(8x,11x,12S)-8,11,12-trihydroxyicosa-5,9,14-trienoate. In simpler terms, this enzyme converts hepoxilin A3 (HxA3), an epoxy-hydroxy eicosanoid, into a trihydroxy fatty acid product. The activity was first purified from rat liver, establishing it as a distinct epoxide hydrolase. Subsequent work demonstrated that mammalian soluble epoxide hydrolase (sEH) is identical to liver hepoxilin hydrolase, unifying a classic lipid-metabolizing enzyme with hepoxilin metabolism. This GO term is therefore central to understanding how epoxy lipids are processed in mammalian tissues. The biological importance of GO:0047977 stems from the fact that hepoxilin A3 is a bioactive mediator formed in several tissues, including the rat aorta and pineal gland. Its conversion by hepoxilin-epoxide hydrolase competes with glutathione conjugation, which produces HxA3-C, a distinct metabolite with its own actions in the central nervous system. Because epoxide hydrolases are broadly involved in lipid metabolism and detoxification, the hepoxilin-epoxide hydrolase activity represents a specific node where eicosanoid signaling intersects with general epoxide processing. For researchers, GO:0047977 provides a precise functional annotation for genes and proteins that hydrolyze hepoxilin A3. It enables comparative studies of epoxide hydrolase family members, supports the interpretation of lipidomic and metabolomic data, and guides the design of CRISPR models to test causality in inflammation, vascular biology, and neurobiology.
hepoxilin-epoxide hydrolase activity At A Glance
| GO ID | GO:0047977 |
|---|---|
| GO term | hepoxilin-epoxide hydrolase activity |
| Ontology | molecular_function |
| Synonym | hepoxilin A3 hydrolase activity; hepoxilin A(3) hydrolase activity; hepoxilin epoxide hydrolase activity; hepoxylin hydrolase activity; (5Z,9E,14Z)-(8xi,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate hydrolase activity |
| Major function | Catalyzes the hydrolysis of hepoxilin A3 to a trihydroxy eicosanoid product |
| Reaction | (5Z,9E,14Z)-(8x,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate + H2O = (5Z,9E,14Z)-(8x,11x,12S)-8,11,12-trihydroxyicosa-5,9,14-trienoate |
| Enzyme class | Epoxide hydrolase |
| Key enzyme | Soluble epoxide hydrolase (sEH, EPHX2) is identical to liver hepoxilin hydrolase |
| Tissue sources | Rat liver, aorta, pineal gland |
What Is GO:0047977?
GO:0047977, hepoxilin-epoxide hydrolase activity, is a molecular function term describing the catalysis of a specific hydrolytic reaction: the conversion of (5Z,9E,14Z)-(8x,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate (hepoxilin A3) plus water into (5Z,9E,14Z)-(8x,11x,12S)-8,11,12-trihydroxyicosa-5,9,14-trienoate. This is an epoxide ring-opening reaction that adds water across the epoxide, yielding a trihydroxy product. The term is synonymous with hepoxilin A3 hydrolase activity, hepoxilin epoxide hydrolase activity, and hepoxylin hydrolase activity. It is classified under the molecular_function aspect of the Gene Ontology.
Why Is hepoxilin-epoxide hydrolase activity Important in Cell Biology?
GO:0047977 is important because it defines a specific enzymatic step in the metabolism of hepoxilin A3, a bioactive eicosanoid. The identity of mammalian soluble epoxide hydrolase with liver hepoxilin hydrolase means that this activity is directly tied to a major drug-metabolizing and lipid-signaling enzyme. Understanding this activity helps explain how epoxy lipids are converted to their dihydroxy or trihydroxy products, which can have distinct biological actions. Because hepoxilin A3 is formed in vascular and neural tissues, the hydrolase activity may influence inflammation, vascular tone, and neurotransmission. The competing glutathione conjugation pathway to HxA3-C further highlights the importance of this enzyme in determining the metabolic fate of hepoxilin A3 in the central nervous system. Finally, epoxide hydrolases as a family are central to lipid mediator biology and xenobiotic detoxification, making GO:0047977 a valuable annotation for functional genomics and drug discovery.
• Defines a specific hydrolytic step in hepoxilin A3 metabolism, distinguishing it from glutathione conjugation.
• Links to soluble epoxide hydrolase (EPHX2), a well-studied enzyme with roles in lipid signaling and detoxification.
• Relevant to vascular biology because hepoxilin A3 is formed by the rat aorta.
• Relevant to neurobiology because hepoxilin A3 is formed and metabolized in the rat pineal gland and central nervous system.
• Provides a functional annotation for epoxide hydrolase family members beyond their generic activities.
• Supports lipidomic and metabolomic interpretation of eicosanoid pathways.
• Enables CRISPR-based causal testing of EPHX2 and related genes in inflammation and vascular disease.
• Helps identify potential drug targets for modulating epoxy lipid signaling.
• Facilitates comparative studies of hepoxilin metabolism across species and tissues.
• Underpins research into the balance between hydrolytic and conjugative hepoxilin A3 clearance.
Molecular Mechanism of hepoxilin-epoxide hydrolase activity
Substrate recognition and binding
In simple terms: The enzyme must first grab hepoxilin A3, the molecule it will modify.
Hepoxilin-epoxide hydrolase activity acts on the substrate (5Z,9E,14Z)-(8x,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate, also known as hepoxilin A3. This epoxy-hydroxy eicosanoid is formed in tissues such as the rat aorta and pineal gland. The enzyme recognizes the epoxide moiety and the hydroxyl group of the substrate, positioning it for hydrolysis. Purification of the enzyme from rat liver demonstrated that this activity is distinct from other epoxide hydrolases and can be assayed with hepoxilin A3 as substrate.
Catalytic hydrolysis of the epoxide
In simple terms: Water is added to the epoxide ring, opening it and converting it to a trihydroxy product.
The catalytic mechanism involves the addition of water across the epoxide, converting hepoxilin A3 to (5Z,9E,14Z)-(8x,11x,12S)-8,11,12-trihydroxyicosa-5,9,14-trienoate. This reaction is characteristic of epoxide hydrolases, which catalyze the hydrolysis of epoxides to diols or related products. The enzyme was originally purified from rat liver based on this hydrolytic activity. The identity of mammalian soluble epoxide hydrolase with liver hepoxilin hydrolase indicates that the same enzyme can hydrolyze both xenobiotic epoxides and hepoxilin A3.
Competition with glutathione conjugation
In simple terms: Instead of being hydrolyzed, hepoxilin A3 can be conjugated to glutathione, creating a different metabolite.
In the rat central nervous system, hepoxilin A3 can be converted to a glutathione conjugate, HxA3-C, which has its own actions. Similarly, the rat aorta metabolizes hepoxilin A3 into HxA3-C. This means that hepoxilin-epoxide hydrolase activity competes with glutathione conjugation for the same substrate, influencing the balance of bioactive metabolites. The pineal gland also forms and metabolizes hepoxilin A3, suggesting that both pathways operate in neuroendocrine tissues.
Enzyme identity and family context
In simple terms: The enzyme that performs this reaction is the same as a well-known soluble epoxide hydrolase.
Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase. This finding connects GO:0047977 to the broader epoxide hydrolase family, which includes enzymes involved in lipid metabolism and detoxification. The soluble epoxide hydrolase is widely expressed and has been studied for its roles in cardiovascular and inflammatory processes. Its dual activity toward hepoxilin A3 and other epoxides makes it a key node in eicosanoid metabolism.
Tissue distribution and physiological context
In simple terms: This activity occurs in tissues like liver, blood vessels, and the pineal gland.
Hepoxilin-epoxide hydrolase activity was first purified from rat liver. Hepoxilin A3, the substrate, is formed by the rat aorta and in the rat pineal gland. The glutathione conjugate HxA3-C is formed in the rat central nervous system. These findings indicate that the enzyme operates in multiple tissues, where it contributes to the local metabolism of hepoxilin A3. The presence of both hydrolytic and conjugative pathways suggests tight regulation of hepoxilin A3 levels in these compartments.
Key Genes Involved in GO:0047977 hepoxilin-epoxide hydrolase activity
The following genes and proteins are directly or indirectly linked to hepoxilin-epoxide hydrolase activity (GO:0047977) based on the verified literature.
| Gene | Major Role | Research Relevance |
|---|---|---|
| EPHX2 | Encodes soluble epoxide hydrolase, identical to liver hepoxilin hydrolase | Central enzyme for GO:0047977; target for KO and point-mutation studies |
| EPHX1 | Microsomal epoxide hydrolase family member | Comparative studies of epoxide hydrolase specificity |
| EPHX3 | Epoxide hydrolase family member | Potential redundancy or substrate overlap |
| EPHX4 | Epoxide hydrolase family member | Family-wide functional annotation |
| GST | Glutathione S-transferase enzymes that form HxA3-C | Competing pathway for hepoxilin A3 metabolism |
| ALOX12 | Lipoxygenase involved in hepoxilin precursor formation | Upstream of hepoxilin A3 synthesis |
| ALOX15 | Lipoxygenase family member | Related eicosanoid pathways |
| CYP450 | Cytochrome P450 epoxygenases generate epoxy lipids | Source of epoxide substrates |
| PLA2 | Phospholipase A2 releases arachidonic acid | Upstream of eicosanoid synthesis |
| COX1 | Cyclooxygenase involved in eicosanoid metabolism | Broader eicosanoid context |
| COX2 | Cyclooxygenase involved in eicosanoid metabolism | Inflammation-related eicosanoid pathways |
| LTA4H | Leukotriene A4 hydrolase, related epoxide metabolism | Comparative epoxide hydrolase biology |
| ABHD | Alpha/beta hydrolase domain proteins | Potential alternative epoxide hydrolases |
| sEH | Soluble epoxide hydrolase protein | Direct enzyme for GO:0047977 |
| HXA3 | Hepoxilin A3, the substrate | Metabolite measured in lipidomic assays |
| HXA3-C | Glutathione conjugate of hepoxilin A3 | Alternative metabolite for pathway balance |
| 12-LOX | 12-lipoxygenase, implicated in hepoxilin formation | Upstream enzyme for substrate supply |
How Is hepoxilin-epoxide hydrolase activity Regulated?
The regulation of hepoxilin-epoxide hydrolase activity is not extensively characterized in the verified literature. However, because mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase, the activity is likely subject to the same regulatory mechanisms that control soluble epoxide hydrolase expression and activity, including transcriptional regulation and post-translational modifications. The balance between hydrolysis and glutathione conjugation of hepoxilin A3 in tissues such as the central nervous system and aorta suggests that substrate availability and competing enzyme activities influence the effective rate of GO:0047977. Further studies are needed to define specific regulatory pathways.
hepoxilin-epoxide hydrolase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| EPHX2 | Cardiovascular and inflammatory diseases | EPHX2 knockout and point-mutation cell models |
| GST | Neuroinflammation and eicosanoid balance | GST overexpression or knockout in neural cells |
| ALOX12 | Hepoxilin A3 synthesis in pineal gland | ALOX12 knockout in pineal-derived cells |
| EPHX1 | Epoxide hydrolase-related detoxification | EPHX1 knockout for comparative studies |
| EPHX4 | Lipid mediator metabolism | EPHX4 overexpression for substrate profiling |
Hepoxilin-epoxide hydrolase activity in vascular biology
Hepoxilin A3 is formed by the rat aorta and is metabolized into HxA3-C, a glutathione conjugate. This suggests that hepoxilin-epoxide hydrolase activity, which competes with conjugation, may influence vascular tone and inflammation. Soluble epoxide hydrolase, the enzyme responsible for this activity, has been studied in cardiovascular contexts. Dysregulation of epoxy lipid metabolism has been linked to hypertension and vascular remodeling, making GO:0047977 a potential node for vascular disease research.
Hepoxilin-epoxide hydrolase activity in the central nervous system
Hepoxilin A3 is formed and metabolized in the rat pineal gland, and its glutathione conjugate HxA3-C has actions in the rat central nervous system. The presence of hepoxilin-epoxide hydrolase activity in neural tissues implies a role in neuroendocrine signaling and possibly in neuroinflammation. Because the enzyme competes with glutathione conjugation, changes in its activity could alter the balance of bioactive hepoxilin metabolites in the brain.
Epoxide hydrolase family and lipid mediator diseases
Epoxide hydrolases are broadly involved in lipid metabolism and detoxification, and their dysregulation has been associated with inflammatory and metabolic diseases. The identity of soluble epoxide hydrolase with hepoxilin hydrolase places GO:0047977 within this larger family. Research into epoxide hydrolase inhibitors often considers their effects on multiple epoxy lipid substrates, including hepoxilin A3. Therefore, understanding GO:0047977 may inform therapeutic strategies targeting epoxide hydrolase in inflammation and cardiovascular disease.
From hepoxilin-epoxide hydrolase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does EPHX2 loss alter hepoxilin A3 hydrolysis? | EPHX2 knockout cell line |
| Does a specific EPHX2 point mutation affect catalytic activity? | Point-mutation knock-in of EPHX2 |
| Can tagged EPHX2 be used to track subcellular localization? | Tagged knock-in of EPHX2 |
| Does EPHX2 overexpression change eicosanoid profiles? | EPHX2 overexpression cell model |
| Which genes modify hepoxilin A3 metabolism? | CRISPR library screening in relevant cell types |
| Does GST loss shift hepoxilin A3 toward hydrolysis? | GST knockout cell line |
How to Study the hepoxilin-epoxide hydrolase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| Enzyme activity assay | Hydrolysis of hepoxilin A3 to trihydroxy product | Validation of GO:0047977 in cell lysates |
| LC-MS/MS lipidomics | Levels of hepoxilin A3 and metabolites | Pathway flux analysis |
| RNA-seq | Expression of EPHX2 and related genes | Transcriptional regulation studies |
| Proteomics | Protein abundance of epoxide hydrolases | Enzyme expression profiling |
| CRISPR knockout | Loss-of-function effects on activity | Causal gene testing |
| CRISPR point mutation | Effect of specific residues on catalysis | Structure-function studies |
| CRISPR knock-in tagging | Subcellular localization of EPHX2 | Imaging and interaction studies |
Enzymatic assays for hepoxilin-epoxide hydrolase activity
Direct measurement of GO:0047977 can be performed using purified enzyme or cell lysates incubated with hepoxilin A3, followed by detection of the trihydroxy product by chromatography or mass spectrometry. The original purification from rat liver used such activity assays. These methods are essential for validating enzyme identity and kinetics.
Lipidomics and metabolomics
Mass spectrometry-based lipidomics can quantify hepoxilin A3 and its metabolites, including the trihydroxy product and HxA3-C. This approach allows researchers to assess the flux through the hydrolytic versus conjugative pathways in cells and tissues. It is particularly useful for studying the balance between GO:0047977 and glutathione conjugation.
Gene expression and proteomics
RNA-seq and proteomics can measure expression of EPHX2 and related epoxide hydrolase genes. These methods help determine whether changes in enzyme abundance correlate with activity. They are also useful for identifying co-regulated genes in eicosanoid pathways.
CRISPR-based functional genomics
CRISPR knockout, point-mutation, and knock-in models enable causal testing of EPHX2 and other candidate genes in hepoxilin metabolism. By comparing wild-type and mutant cells, researchers can determine whether a specific gene is required for GO:0047977 activity. Library screening can identify additional modifiers of the pathway.
How CRISPR Can Be Used to Study GO:0047977 hepoxilin-epoxide hydrolase activity
Knockout
CRISPR knockout of EPHX2 can abolish hepoxilin-epoxide hydrolase activity, allowing researchers to test its contribution to hepoxilin A3 metabolism. Because soluble epoxide hydrolase is identical to liver hepoxilin hydrolase, EPHX2 knockout models are directly relevant. Knockout cells can be used in lipidomic assays to measure accumulation of hepoxilin A3 or shunting to HxA3-C.
Point Mutation
Point mutations in EPHX2 can be introduced to dissect catalytic residues or regulatory sites. Such models help determine which amino acids are essential for GO:0047977 activity. They are particularly useful when a complete knockout is lethal or when studying subtle changes in enzyme kinetics.
Knock-in
Knock-in of tagged EPHX2 (e.g., GFP or FLAG) enables visualization and immunoprecipitation of the enzyme. This approach can reveal subcellular localization and interaction partners. It also allows correlation of enzyme abundance with activity in live cells.
Overexpression
Overexpression of EPHX2 or other epoxide hydrolases can increase hepoxilin-epoxide hydrolase activity, providing a gain-of-function system. This is useful for testing whether increased hydrolysis alters eicosanoid profiles or cellular phenotypes. Overexpression models complement knockout studies to establish causality.
How EDITGENE Supports hepoxilin-epoxide hydrolase activity Research
Researchers studying hepoxilin-epoxide hydrolase activity-related genes often need to determine whether a candidate gene is causally involved in the hydrolysis of hepoxilin A3 or in related eicosanoid pathways. CRISPR-based models provide a precise way to manipulate EPHX2 and other candidate genes, enabling functional validation in relevant cell types.
Contact EDITGENE today to design your custom CRISPR model for hepoxilin-epoxide hydrolase activity research.
Frequently Asked Questions About hepoxilin-epoxide hydrolase activity
What is hepoxilin-epoxide hydrolase activity?
Hepoxilin-epoxide hydrolase activity (GO:0047977) is a molecular function that catalyzes the hydrolysis of hepoxilin A3 to a trihydroxy eicosanoid product.
What is the GO ID for hepoxilin-epoxide hydrolase activity?
The Gene Ontology ID is GO:0047977.
Which enzyme carries out hepoxilin-epoxide hydrolase activity?
Mammalian soluble epoxide hydrolase (EPHX2) is identical to liver hepoxilin hydrolase, which carries out this activity.
What reaction does GO:0047977 catalyze?
It catalyzes the conversion of (5Z,9E,14Z)-(8x,11R,12S)-11,12-epoxy-8-hydroxyicosa-5,9,14-trienoate plus water to (5Z,9E,14Z)-(8x,11x,12S)-8,11,12-trihydroxyicosa-5,9,14-trienoate.
What genes are involved in hepoxilin-epoxide hydrolase activity?
EPHX2 is the primary gene, and other epoxide hydrolase family members such as EPHX1, EPHX3, and EPHX4 may also be relevant.
Where is hepoxilin-epoxide hydrolase activity found?
It was first purified from rat liver and is relevant in tissues where hepoxilin A3 is formed, such as the rat aorta and pineal gland.
How is hepoxilin A3 metabolized?
Hepoxilin A3 can be hydrolyzed by hepoxilin-epoxide hydrolase or conjugated with glutathione to form HxA3-C.
What diseases are linked to hepoxilin-epoxide hydrolase activity?
It has been studied in the context of vascular biology, neuroinflammation, and eicosanoid-related diseases.
How can I study hepoxilin-epoxide hydrolase activity in the lab?
Enzymatic assays, lipidomics, and CRISPR knockout or overexpression models of EPHX2 are commonly used.
Does EDITGENE provide CRISPR models for hepoxilin-epoxide hydrolase research?
Yes, EDITGENE offers knockout, point-mutation, knock-in, overexpression, and library screening services for EPHX2 and related genes.
Conclusion
GO:0047977, hepoxilin-epoxide hydrolase activity, defines a specific hydrolytic step in the metabolism of the bioactive eicosanoid hepoxilin A3. The identity of mammalian soluble epoxide hydrolase with liver hepoxilin hydrolase provides a direct link between this activity and a well-studied enzyme, while tissue-specific studies in liver, aorta, and pineal gland highlight its physiological relevance. The competition with glutathione conjugation to form HxA3-C further underscores the importance of this activity in determining the fate of hepoxilin A3 in the central nervous system and vasculature. For researchers, GO:0047977 offers a precise annotation for functional genomics and lipidomics. CRISPR-based models of EPHX2 and related genes enable causal testing of this activity in inflammation, vascular biology, and neurobiology. EDITGENE provides comprehensive services to support such studies, from knockout and point-mutation cell lines to library screening and bioinformatics.
References
- 1. Cronin A et al.. 2011. Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase.. J Lipid Res 52(4):712-9 PMID: 21217101
- 2. Pace-Asciak CR et al.. 1989. Purification of hepoxilin epoxide hydrolase from rat liver.. J Biol Chem 264(16):9310-3 PMID: 2722835
- 3. Pace-Asciak CR et al.. 1990. A glutathione conjugate of hepoxilin A3: formation and action in the rat central nervous system.. Proc Natl Acad Sci U S A 87(8):3037-41 PMID: 2326264
- 4. Newman JW et al.. 2005. Epoxide hydrolases: their roles and interactions with lipid metabolism.. Prog Lipid Res 44(1):1-51 PMID: 15748653
- 5. Laneuville O et al.. 1991. Hepoxilin A3 (HxA3) is formed by the rat aorta and is metabolized into HxA3-C, a glutathione conjugate.. Biochim Biophys Acta 1084(1):60-8 PMID: 1675872
- 6. Reynaud D et al.. 1994. Formation, metabolism, and action of hepoxilin A3 in the rat pineal gland.. J Neurochem 62(1):126-33 PMID: 8263512
