GO:0000247 C-8 sterol isomerase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0000247 C-8 sterol isomerase activity catalyzes the conversion of fecosterol to episterol, a step in the sterol biosynthesis pathway.
• The enzyme is also known as delta-8-delta-7 sterol isomerase and is encoded by the ERG2 gene in yeast and the EBP gene in mammals.
• C-8 sterol isomerase activity is essential for the production of ergosterol in fungi and cholesterol in mammals.
• Mutations in the human EBP gene cause X-linked dominant chondrodysplasia punctata 2 (CDPX2), a developmental disorder.
• The enzyme is a target for antifungal drugs and has been implicated in cancer cell proliferation.
• Research methods include enzyme assays, CRISPR knockout models, and structural studies to understand its catalytic mechanism.
Description
C-8 sterol isomerase activity (GO:0000247) is a molecular function that catalyzes the isomerization of fecosterol to episterol, a critical step in the sterol biosynthesis pathway. This enzymatic activity is essential for the production of major sterols such as ergosterol in fungi and cholesterol in mammals. The enzyme is known as delta-8-delta-7 sterol isomerase and is encoded by the ERG2 gene in yeast and the EBP gene in humans. Researchers study this activity to understand sterol metabolism, membrane function, and the development of drugs targeting sterol biosynthesis. Defects in this enzyme lead to developmental disorders and are explored in cancer research.
C-8 sterol isomerase activity At A Glance
| GO ID | GO:0000247 |
|---|---|
| GO term | C-8 sterol isomerase activity |
| Ontology | molecular_function |
| Synonym | delta-8-delta-7 sterol isomerase activity |
| Definition | Catalysis of the reaction: fecosterol = episterol. |
| Major function | Isomerization of fecosterol to episterol in sterol biosynthesis |
| EC number | 5.3.3.5 |
| Pathway | Sterol biosynthesis (KEGG: map00100) |
| Cellular location | Endoplasmic reticulum membrane |
What Is GO:0000247?
C-8 sterol isomerase activity is defined as the catalysis of the reaction: fecosterol = episterol. This means the enzyme rearranges the double bond in the sterol B-ring from the C-8(9) position to the C-7(8) position, converting fecosterol to episterol. This isomerization is a key step in the post-squalene portion of sterol biosynthesis.
Why Is C-8 sterol isomerase activity Important in Cell Biology?
C-8 sterol isomerase activity is crucial because it is a committed step in the biosynthesis of sterols, which are essential components of cell membranes and precursors for steroid hormones and bile acids. In fungi, this activity is required for ergosterol production, making it a target for antifungal agents. In humans, impaired activity due to mutations in the EBP gene causes CDPX2, a disorder affecting bone and cartilage development. Additionally, the enzyme is implicated in cancer, as its inhibition affects cell proliferation. Understanding this activity provides insights into sterol-related diseases and potential therapeutic interventions.
• Essential for ergosterol biosynthesis in fungi, a target for antifungal drugs.
• Required for cholesterol biosynthesis in mammals, impacting membrane integrity and hormone production.
• Mutations in the EBP gene cause X-linked chondrodysplasia punctata 2.
• Involved in cancer cell proliferation and potential anticancer targets.
• Key enzyme in the sterol biosynthesis pathway, studied for evolutionary conservation.
• Subject to inhibition by drugs like tamoxifen, affecting sterol metabolism.
• Plays a role in plant sterol biosynthesis, with homologs in Arabidopsis and Zea mays.
• Its activity can be modulated by synthetic ligands, offering pharmacological tools.
• Important for understanding sterol-related metabolic disorders.
• Provides a model for studying enzyme mechanism and structure-function relationships.
Mechanism, Genes and Research Methods
Substrate Binding and Isomerization
In simple terms: The enzyme grabs fecosterol and rearranges its double bond to make episterol.
C-8 sterol isomerase binds fecosterol, a sterol with a double bond at C-8(9). Through a protonation-deprotonation mechanism, the enzyme moves the double bond to C-7(8), forming episterol. This isomerization is essential for further sterol modifications. The reaction is stereospecific and requires no cofactors.
Enzyme Structure and Active Site
In simple terms: The enzyme has a specific pocket where the reaction happens, with key amino acids that help move the double bond.
The enzyme is an integral membrane protein of the endoplasmic reticulum. Structural studies and mutagenesis have identified essential amino acid residues, such as histidine and aspartate, in the active site that facilitate the isomerization. The mammalian enzyme, emopamil-binding protein (EBP), shares functional homology with fungal and plant enzymes.
Role in Sterol Biosynthesis Pathway
In simple terms: This step is like a checkpoint in the assembly line that makes cholesterol or ergosterol.
In the sterol biosynthesis pathway, C-8 sterol isomerase acts after the formation of fecosterol. The product, episterol, is subsequently converted to ergosterol in fungi or cholesterol in mammals. This step is regulated by feedback mechanisms and can be inhibited by compounds like tamoxifen.
Regulation and Inhibition
In simple terms: The enzyme's activity can be turned up or down by other molecules, affecting how much sterol is made.
C-8 sterol isomerase activity is regulated at the transcriptional level and by feedback inhibition from downstream sterols. Pharmacological inhibitors, such as tamoxifen and synthetic piperazine derivatives, can block the enzyme, leading to accumulation of fecosterol and reduced sterol synthesis. These inhibitors have antiproliferative effects in cancer cells.
Key Genes Involved in GO:0000247 C-8 sterol isomerase activity
The following genes encode proteins with C-8 sterol isomerase activity or are directly involved in its function and regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| ERG2 (yeast) | Encodes C-8 sterol isomerase in Saccharomyces cerevisiae | Model for antifungal drug discovery and sterol metabolism |
| EBP (human) | Encodes emopamil-binding protein with C-8 sterol isomerase activity | Mutations cause CDPX2; target for cancer and cholesterol research |
| EBP (mouse) | Ortholog of human EBP | Studied for developmental and neurological roles |
| AtEBP (Arabidopsis) | Plant C-8,7 sterol isomerase | Model for plant sterol biosynthesis and development |
| ZmEBP (Zea mays) | Maize sterol 8,7-isomerase | Studied for essential amino acid residues and functional homology |
| SC5D | Sterol-C5-desaturase, acts downstream of C-8 isomerase | Related enzyme in cholesterol biosynthesis |
| DHCR7 | 7-dehydrocholesterol reductase, downstream enzyme | Defects cause Smith-Lemli-Opitz syndrome |
| DHCR24 | 24-dehydrocholesterol reductase, involved in sterol pathway | Linked to desmosterolosis |
| FDFT1 | Squalene synthase, upstream of sterol isomerase | Target for cholesterol-lowering drugs |
| SQLE | Squalene monooxygenase, upstream enzyme | Regulated in sterol biosynthesis |
| LSS | Lanosterol synthase, upstream enzyme | Catalyzes cyclization of squalene oxide |
| CYP51 | Lanosterol 14α-demethylase, upstream enzyme | Target for antifungal azoles |
| ERG3 (yeast) | C-5 sterol desaturase, downstream of ERG2 | Involved in ergosterol biosynthesis |
| ERG5 (yeast) | C-22 sterol desaturase, downstream of ERG2 | Ergosterol pathway enzyme |
| ERG6 (yeast) | Sterol C-24 methyltransferase, upstream of ERG2 | Modifies sterol intermediates |
| TM7SF2 | Delta(14)-sterol reductase, related sterol reductase | Involved in cholesterol biosynthesis |
| NSDHL | Sterol-4-alpha-carboxylate 3-dehydrogenase, sterol pathway | Mutations cause CHILD syndrome |
| MVD | Mevalonate diphosphate decarboxylase, upstream in mevalonate pathway | Provides precursors for sterol synthesis |
How Is C-8 sterol isomerase activity Regulated?
C-8 sterol isomerase activity is regulated primarily at the transcriptional level in response to sterol levels. In yeast, ERG2 expression is induced under anaerobic conditions and repressed by ergosterol. In mammals, EBP expression is controlled by SREBP (sterol regulatory element-binding protein) transcription factors, which sense cholesterol levels. Additionally, the enzyme can be inhibited by pharmacological agents such as tamoxifen, which differentially inhibits sterol delta 8-isomerase compared to other lanosterol-converting enzymes. Synthetic ligands like piperazine derivatives have been developed as selective inhibitors, affecting cell proliferation.
C-8 sterol isomerase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| EBP | Chondrodysplasia punctata 2 (CDPX2) | EBP knockout mouse, patient-derived fibroblasts |
| EBP | Cancer cell proliferation and drug resistance | Cancer cell lines with EBP overexpression or knockout |
| ERG2 | Fungal ergosterol biosynthesis and antifungal resistance | Yeast ERG2 deletion strains |
| EBP | Sterol metabolism disorders | CRISPR knock-in of patient mutations in cell lines |
| AtEBP | Plant sterol biosynthesis and development | Arabidopsis T-DNA insertion mutants |
Chondrodysplasia Punctata 2 (CDPX2)
Mutations in the EBP gene, which encodes C-8 sterol isomerase, cause X-linked dominant chondrodysplasia punctata 2 (CDPX2), also known as Conradi-Hünermann-Happle syndrome. This disorder is characterized by skeletal abnormalities, stippled epiphyses, and skin lesions. The mutations lead to reduced enzyme activity and accumulation of abnormal sterols, affecting bone and cartilage development.
Cancer
C-8 sterol isomerase activity has been implicated in cancer cell proliferation. Inhibitors of the enzyme, such as piperazine derivatives, exhibit antiproliferative activity in cancer cell lines and also inhibit P-glycoprotein, a multidrug resistance transporter. This suggests that targeting this enzyme could be a strategy for cancer therapy, particularly in drug-resistant cancers.
Antifungal Drug Target
In fungi, C-8 sterol isomerase is essential for ergosterol biosynthesis, which is a key component of fungal cell membranes. Disruption of the ERG2 gene leads to ergosterol auxotrophy and increased sensitivity to antifungal agents. Therefore, the enzyme is a potential target for antifungal drug development, especially against pathogenic fungi.
From C-8 sterol isomerase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of C-8 sterol isomerase loss on sterol composition? | CRISPR knockout of EBP in HeLa or HEK293 cells |
| How do disease-causing mutations affect enzyme activity? | Point mutation knock-in of EBP mutations (e.g., R147H) in cell lines |
| Can we tag the enzyme to study its localization? | Knock-in of fluorescent protein tag (e.g., GFP) at the EBP locus |
| What is the effect of enzyme overexpression on cholesterol synthesis? | Overexpression of EBP in mammalian cells |
| How does the enzyme interact with inhibitors? | In vitro enzyme assays with purified recombinant EBP |
| What genes are regulated by C-8 sterol isomerase activity? | CRISPR knockout followed by RNA-seq in yeast or mammalian cells |
How to Study the C-8 sterol isomerase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| GC-MS sterol profiling | Levels of fecosterol, episterol, and other sterols | Assessing enzyme activity in cells or tissues |
| Enzyme assay with radiolabeled substrate | Conversion of fecosterol to episterol | In vitro kinetic studies |
| CRISPR knockout | Loss of enzyme function | Studying essentiality and metabolic consequences |
| Site-directed mutagenesis | Effect of specific amino acid changes on activity | Identifying catalytic residues |
| Western blot | Protein expression levels | Validating knockout or overexpression |
| RNA-seq | Transcriptional changes upon enzyme loss | Identifying regulated pathways |
| Immunofluorescence | Subcellular localization | Confirming endoplasmic reticulum localization |
| Drug inhibition assays | IC50 of inhibitors | Screening for potential therapeutics |
Enzyme Activity Assays
C-8 sterol isomerase activity can be measured using in vitro assays with radiolabeled fecosterol or by monitoring the conversion of fecosterol to episterol via gas chromatography-mass spectrometry (GC-MS). Purified enzyme or cell lysates are incubated with substrate, and products are analyzed.
CRISPR-Cas9 Knockout Studies
CRISPR-Cas9 can be used to generate knockout cell lines or organisms to study the loss of C-8 sterol isomerase activity. For example, ERG2 knockout yeast strains are viable only with ergosterol supplementation, demonstrating the enzyme's essential role. In mammalian cells, EBP knockout leads to cholesterol auxotrophy and developmental defects.
Structural and Mutagenesis Studies
Site-directed mutagenesis has identified essential amino acid residues in the active site of C-8 sterol isomerase. For instance, in Zea mays, conserved histidine and aspartate residues are critical for catalysis. Structural models based on homology to other isomerases provide insights into the reaction mechanism.
Pharmacological Inhibition
Small molecule inhibitors, such as tamoxifen and piperazine derivatives, are used to inhibit C-8 sterol isomerase activity in cells. These compounds can be tested for their effects on sterol synthesis and cell proliferation, providing a way to study the enzyme's role in disease.
How CRISPR Can Be Used to Study GO:0000247 C-8 sterol isomerase activity
Knockout
CRISPR-Cas9 knockout of EBP or ERG2 generates cell lines or organisms lacking C-8 sterol isomerase activity. These models are used to study the metabolic consequences, such as sterol auxotrophy and accumulation of fecosterol, and to validate the enzyme as a drug target.
Point Mutation
Point mutations identified in CDPX2 patients (e.g., in EBP) can be introduced into cell lines using CRISPR-Cas9 homology-directed repair. These models help determine how specific mutations affect enzyme activity and contribute to disease phenotypes.
Knock-in
Knock-in of tags (e.g., GFP or FLAG) at the endogenous EBP locus allows for real-time tracking of enzyme localization and interaction partners. This approach is valuable for studying the enzyme's dynamics and regulation in live cells.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression can be used to increase C-8 sterol isomerase levels. Overexpression models are useful for studying the effects of excess enzyme activity on sterol synthesis and cell proliferation, and for drug screening.
How EDITGENE Supports C-8 sterol isomerase activity Research
Researchers studying C-8 sterol isomerase activity-related genes often need to determine whether a candidate gene is causally involved in sterol metabolism, disease development, or drug response. EDITGENE provides comprehensive CRISPR-based services to create precise cellular models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for C-8 sterol isomerase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| SREBF2 Knockout HEK293 Cell Line | EDJ-KQ1125 | Human | 6721 | Details Get a Quote |
| SREBF2 Knockout A-549 Cell Line | EDJ-KQ20321 | Human | 6721 | Details Get a Quote |
| SREBF2 Knockout HCT 116 Cell Line | EDJ-KQ20322 | Human | 6721 | Details Get a Quote |
| SREBF2 Knockout HeLa Cell Line | EDJ-KQ20323 | Human | 6721 | Details Get a Quote |
| EBP Knockout HEK293 Cell Line | EDJ-KQ51000 | Human | 10682 | Details Get a Quote |
| EBP Knockout HeLa Cell Line | EDJ-KQ55461 | Human | 10682 | Details Get a Quote |
| EBP Knockout A-549 Cell Line | EDJ-KQ63946 | Human | 10682 | Details Get a Quote |
| EBP Knockout HCT 116 Cell Line | EDJ-KQ72402 | Human | 10682 | Details Get a Quote |
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Frequently Asked Questions About C-8 sterol isomerase activity
What is C-8 sterol isomerase activity?
C-8 sterol isomerase activity (GO:0000247) is the catalysis of the reaction converting fecosterol to episterol, a step in sterol biosynthesis.
What genes are involved in C-8 sterol isomerase activity?
The main genes are ERG2 in yeast and EBP in humans, with homologs in plants like Arabidopsis and Zea mays.
What diseases are associated with C-8 sterol isomerase deficiency?
Mutations in the EBP gene cause X-linked chondrodysplasia punctata 2 (CDPX2), a developmental disorder.
How is C-8 sterol isomerase activity measured?
It can be measured using enzyme assays with radiolabeled substrate or GC-MS to detect fecosterol and episterol levels.
What is the role of C-8 sterol isomerase in cholesterol synthesis?
It catalyzes a step in the cholesterol biosynthesis pathway, converting fecosterol to episterol, which is further modified to cholesterol.
Can C-8 sterol isomerase be targeted for antifungal therapy?
Yes, because it is essential for ergosterol biosynthesis in fungi, inhibitors could serve as antifungal drugs.
What are the inhibitors of C-8 sterol isomerase?
Tamoxifen and synthetic piperazine derivatives have been shown to inhibit the enzyme.
Is C-8 sterol isomerase involved in cancer?
Yes, its inhibition has antiproliferative effects in cancer cells, and it is being explored as a target.
What model organisms are used to study C-8 sterol isomerase?
Saccharomyces cerevisiae (ERG2), Arabidopsis thaliana, Zea mays, and mammalian cell lines are commonly used.
How can CRISPR be used to study C-8 sterol isomerase?
CRISPR can create knockouts, point mutations, or tagged knock-ins of EBP or ERG2 to study function and disease mechanisms.
Conclusion
C-8 sterol isomerase activity (GO:0000247) is a critical enzymatic step in sterol biosynthesis, essential for membrane integrity and hormone production. Its role in fungal ergosterol synthesis and human cholesterol synthesis makes it a target for antifungal and anticancer therapies. Mutations in the EBP gene cause CDPX2, highlighting its importance in development. Continued research using CRISPR models and biochemical assays will further elucidate its mechanism and therapeutic potential.
References
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- 2. Grebenok RJ et al.. 1998. Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein.. Plant Mol Biol 38(5):807-15 PMID: 9862498
- 3. Abate C et al.. 2011. 1-Cyclohexyl-4-(4-arylcyclohexyl)piperazines: Mixed σ and human Δ(8)-Δ(7) sterol isomerase ligands with antiproliferative and P-glycoprotein inhibitory activity.. ChemMedChem 6(1):73-80 PMID: 21069657
- 4. Kang MK et al.. 1995. Cholesterol biosynthesis from lanosterol: regulation and purification of rat hepatic sterol 8-isomerase.. J Biochem 117(4):819-23 PMID: 7592544
- 5. Berardi F et al.. 2008. Novel 4-(4-aryl)cyclohexyl-1-(2-pyridyl)piperazines as Delta(8)-Delta(7) sterol isomerase (emopamil binding protein) selective ligands with antiproliferative activity.. J Med Chem 51(23):7523-31 PMID: 19053780
- 6. Rahier A et al.. 2008. Identification of essential amino acid residues in a sterol 8,7-isomerase from Zea mays reveals functional homology and diversity with the isomerases of animal and fungal origin.. Biochem J 414(2):247-59 PMID: 18459942
- 7. Cho SY et al.. 1998. Cholesterol biosynthesis from lanosterol: differential inhibition of sterol delta 8-isomerase and other lanosterol-converting enzymes by tamoxifen.. Mol Cells 8(2):233-9 PMID: 9638657
- 8. Moebius FF et al.. 2003. Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol delta8-delta7 isomerase activity.. Biochem J 374(Pt 1):229-37 PMID: 12760743
