GO:0047750 cholestenol delta-isomerase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0047750 (cholestenol delta-isomerase activity) catalyzes the isomerization of 5-alpha-cholest-7-en-3-beta-ol to 5-alpha-cholest-8-en-3-beta-ol, a key step in cholesterol biosynthesis.
• The enzyme is also known as emopamil-binding protein (EBP) and exhibits delta8-delta7 sterol isomerase activity in yeast and mammals.
• Human EBP is a membrane protein that binds structurally diverse neuroprotective agents and is linked to X-linked dominant chondrodysplasia punctata 2 (CDPX2).
• Structural studies reveal that human sterol isomerase has a distinct fold with a deep hydrophobic cavity for substrate binding and multidrug recognition.
• A related EBP-like protein lacks sterol delta8-delta7 isomerase activity, highlighting the specificity of GO:0047750.
• Thermostabilization of a fungal delta8-delta7 sterol isomerase by consensus mutation provides insights into membrane protein stability and function.
Description
Cholestenol delta-isomerase activity (GO:0047750) is a molecular function that catalyzes the conversion of 5-alpha-cholest-7-en-3-beta-ol to 5-alpha-cholest-8-en-3-beta-ol, a critical isomerization step in the cholesterol biosynthesis pathway. This activity is essential for maintaining sterol homeostasis in eukaryotic cells and is carried out by the enzyme emopamil-binding protein (EBP), which also exhibits delta8-delta7 sterol isomerase activity. The enzyme is a membrane-bound protein localized to the endoplasmic reticulum and is characterized by its ability to bind a variety of structurally diverse pharmacological agents, including neuroprotective drugs. Researchers study GO:0047750 to understand cholesterol metabolism, membrane biogenesis, and the molecular basis of disorders such as X-linked dominant chondrodysplasia punctata 2 (CDPX2). The structural and functional characterization of this enzyme has been advanced by crystallographic studies that reveal a unique fold with a deep hydrophobic cavity, providing a framework for understanding substrate specificity and drug interactions. Additionally, the enzyme's activity is conserved across species, as demonstrated by the cloning of an EBP-like protein that lacks isomerase activity, underscoring the evolutionary and functional significance of this specific molecular function.
cholestenol delta-isomerase activity At A Glance
| GO ID | GO:0047750 |
|---|---|
| GO term | cholestenol delta-isomerase activity |
| Ontology | molecular_function |
| Synonym | cholestenol D-isomerase activity; delta7-cholestenol delta7-delta8-isomerase activity |
| Major function | Catalyzes the isomerization of 5-alpha-cholest-7-en-3-beta-ol to 5-alpha-cholest-8-en-3-beta-ol |
| Enzyme | Emopamil-binding protein (EBP) |
| Localization | Endoplasmic reticulum membrane |
| Pathway | Cholesterol biosynthesis |
| Related activity | Delta8-delta7 sterol isomerase activity |
What Is GO:0047750?
Cholestenol delta-isomerase activity (GO:0047750) is defined as the catalysis of the reaction: 5-alpha-cholest-7-en-3-beta-ol = 5-alpha-cholest-8-en-3-beta-ol. In other words, it is the enzyme activity that rearranges the double bond in the sterol B-ring from the delta-7 position to the delta-8 position, converting cholest-7-en-3-beta-ol to cholest-8-en-3-beta-ol. This isomerization is a non-oxidative rearrangement that is essential for the proper synthesis of cholesterol and other sterols.
Why Is cholestenol delta-isomerase activity Important in Cell Biology?
Cholestenol delta-isomerase activity is a critical enzymatic step in the cholesterol biosynthesis pathway, and its dysfunction is directly linked to human disease. Mutations in the gene encoding this activity, EBP, cause X-linked dominant chondrodysplasia punctata 2 (CDPX2), a disorder characterized by skeletal abnormalities and skin lesions. The enzyme is also a target for neuroprotective drugs and is involved in multidrug recognition, making it a subject of pharmacological interest. Understanding this activity at the molecular level is essential for developing therapies for cholesterol-related disorders and for deciphering the mechanisms of membrane protein function.
• Essential for cholesterol biosynthesis and membrane integrity.
• Mutations in EBP cause X-linked dominant chondrodysplasia punctata 2 (CDPX2).
• Target of neuroprotective agents and multidrug recognition.
• Provides a model for studying membrane protein structure and stability.
• Conserved across species, from yeast to humans.
• Related to delta8-delta7 sterol isomerase activity in yeast.
• Involved in sterol homeostasis and lipid metabolism.
• Potential target for antiproliferative and P-glycoprotein inhibitory compounds.
• Structural insights aid in understanding enzyme mechanism and drug design.
• Thermostabilization studies offer clues for stabilizing membrane proteins.
What Happens During cholestenol delta-isomerase activity?
Substrate Binding and Recognition
In simple terms: The enzyme grabs the sterol molecule and holds it in place.
The enzyme emopamil-binding protein (EBP) binds the substrate 5-alpha-cholest-7-en-3-beta-ol within a deep hydrophobic cavity, as revealed by the crystal structure of human sterol isomerase. This binding is specific and involves interactions with conserved residues that position the sterol for catalysis.
Catalytic Isomerization
In simple terms: The enzyme rearranges the double bond in the sterol ring.
The isomerization reaction proceeds through a mechanism that likely involves a carbocation intermediate, facilitated by acidic residues in the active site. The enzyme converts the delta-7 double bond to a delta-8 double bond, yielding 5-alpha-cholest-8-en-3-beta-ol. This step is essential for the subsequent conversion of sterols in the cholesterol biosynthesis pathway.
Product Release
In simple terms: The enzyme lets go of the modified sterol.
After isomerization, the product 5-alpha-cholest-8-en-3-beta-ol is released from the active site, allowing the enzyme to participate in further catalytic cycles. The release may be facilitated by conformational changes in the enzyme, as suggested by structural studies.
Regulation by Membrane Environment
In simple terms: The membrane around the enzyme affects its activity.
As a membrane protein, EBP's activity is influenced by the lipid bilayer composition. Studies on a fungal delta8-delta7 sterol isomerase have shown that consensus mutations can thermostabilize the protein, indicating that membrane interactions are critical for stability and function.
Key Genes Involved in GO:0047750 cholestenol delta-isomerase activity
The following genes and proteins are directly associated with cholestenol delta-isomerase activity (GO:0047750) or its related sterol isomerase functions.
| Gene | Major Role | Research Relevance |
|---|---|---|
| EBP | Encodes emopamil-binding protein, the enzyme with cholestenol delta-isomerase activity | Mutations cause CDPX2; target for neuroprotective drugs |
| EBPL | EBP-like protein that lacks sterol delta8-delta7 isomerase activity | Provides a negative control for studying isomerase specificity |
| SC5D | Sterol-C5-desaturase, acts downstream in cholesterol biosynthesis | Related to sterol metabolism but distinct from GO:0047750 |
| DHCR7 | 7-dehydrocholesterol reductase, involved in cholesterol synthesis | Defects cause Smith-Lemli-Opitz syndrome; interacts with isomerase pathway |
| DHCR24 | 24-dehydrocholesterol reductase, involved in cholesterol biosynthesis | Related to sterol homeostasis |
| NSDHL | NAD(P) dependent steroid dehydrogenase-like, cholesterol biosynthesis | Mutations cause CHILD syndrome; related pathway |
| TM7SF2 | Transmembrane 7 superfamily member 2, sterol reductase | Cholesterol biosynthesis enzyme |
| CYP51A1 | Lanosterol 14-alpha demethylase | Upstream of isomerase in cholesterol synthesis |
| SQLE | Squalene epoxidase | Cholesterol biosynthesis |
| HMGCR | HMG-CoA reductase, rate-limiting enzyme | Cholesterol biosynthesis |
| LSS | Lanosterol synthase | Cholesterol biosynthesis |
| FDFT1 | Farnesyl-diphosphate farnesyltransferase 1 | Cholesterol biosynthesis |
| MVK | Mevalonate kinase | Cholesterol biosynthesis |
| PMVK | Phosphomevalonate kinase | Cholesterol biosynthesis |
| MVD | Mevalonate decarboxylase | Cholesterol biosynthesis |
| IDI1 | Isopentenyl-diphosphate delta isomerase 1 | Cholesterol biosynthesis |
| FDPS | Farnesyl diphosphate synthase | Cholesterol biosynthesis |
How Is cholestenol delta-isomerase activity Regulated?
The activity of cholestenol delta-isomerase is regulated at multiple levels. Transcription of the EBP gene is controlled by sterol regulatory element-binding proteins (SREBPs) in response to cellular cholesterol levels, as part of the feedback regulation of cholesterol biosynthesis. Additionally, the enzyme's activity may be modulated by post-translational modifications and membrane lipid composition. Pharmacological agents such as 1-cyclohexyl-4-(4-arylcyclohexyl)piperazines can bind to the enzyme and inhibit its activity, suggesting that small molecules can regulate its function.
cholestenol delta-isomerase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| EBP | CDPX2 (chondrodysplasia punctata 2) | EBP knockout or point-mutation cell lines; patient-derived fibroblasts |
| EBP | Cancer cell proliferation | Cancer cell lines with EBP overexpression or knockout; antiproliferative assays |
| EBP | Neuroprotection | Neuronal cell lines treated with neuroprotective agents; EBP binding assays |
| EBPL | Lack of isomerase activity | EBPL overexpression in sterol isomerase-deficient yeast |
| Fungal delta8-delta7 sterol isomerase | Membrane protein stability | Thermostabilized mutants in fungal cells |
X-linked Dominant Chondrodysplasia Punctata 2 (CDPX2)
Mutations in the EBP gene, which encodes the enzyme with cholestenol delta-isomerase activity, 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 loss of isomerase activity leads to accumulation of abnormal sterols and disrupted cholesterol biosynthesis.
Cancer and Cell Proliferation
Sterol isomerase activity has been implicated in cell proliferation, and inhibitors such as 1-cyclohexyl-4-(4-arylcyclohexyl)piperazines exhibit antiproliferative activity and P-glycoprotein inhibitory effects. These compounds bind to the enzyme and may interfere with cholesterol metabolism in cancer cells, suggesting a potential role in oncology.
Neurodegeneration and Neuroprotection
Emopamil-binding protein was originally identified as a target for neuroprotective agents, and its ability to bind structurally diverse drugs suggests a role in neuroprotection. The enzyme's activity may influence membrane sterol composition, which is critical for neuronal function and survival.
From cholestenol delta-isomerase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does EBP loss affect cholesterol biosynthesis? | EBP knockout HEK293 or HepG2 cells |
| What is the effect of CDPX2-associated mutations on isomerase activity? | Point-mutation knock-in of EBP mutations in cell lines |
| Can EBP be tagged for localization studies? | Knock-in of fluorescent tags (e.g., GFP) at the EBP locus |
| Does EBP overexpression alter sterol profiles? | EBP overexpression in Chinese hamster ovary (CHO) cells |
| Can EBP rescue yeast lacking sterol isomerase? | Expression of human EBP in yeast erg2 mutants |
| What is the structural basis of drug binding? | Crystallography of purified EBP with ligands |
How to Study the cholestenol delta-isomerase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| HPLC/GC-MS | Conversion of substrate to product | Enzyme activity assays |
| X-ray crystallography | Three-dimensional structure | Structural basis of catalysis and drug binding |
| Site-directed mutagenesis | Effect of mutations on activity | Structure-function studies |
| RNA-seq | Gene expression levels | Transcriptional regulation |
| Western blot | Protein expression | Overexpression or knockout validation |
| Immunofluorescence | Subcellular localization | Endoplasmic reticulum localization |
| Isothermal titration calorimetry | Binding affinity | Drug-enzyme interactions |
Enzymatic Activity Assays
Cholestenol delta-isomerase activity can be measured using radiolabeled substrate (5-alpha-cholest-7-en-3-beta-ol) and monitoring its conversion to the delta-8 isomer by HPLC or GC-MS. This method is used to assess enzyme kinetics and inhibition.
Structural Biology
X-ray crystallography and cryo-EM have been used to determine the structure of human sterol isomerase, revealing a deep hydrophobic cavity and the basis for multidrug recognition. These techniques are essential for understanding the catalytic mechanism and designing inhibitors.
Mutagenesis and Thermostabilization
Site-directed mutagenesis and consensus mutation approaches have been applied to a fungal delta8-delta7 sterol isomerase to improve thermostability, providing insights into membrane protein stability and function.
Gene Expression Analysis
Quantitative RT-PCR and RNA-seq can be used to measure EBP mRNA levels in response to cholesterol levels or pharmacological agents. This helps in understanding transcriptional regulation.
How CRISPR Can Be Used to Study GO:0047750 cholestenol delta-isomerase activity
Knockout
CRISPR-Cas9 knockout of EBP can be used to create cell models lacking cholestenol delta-isomerase activity. These models are valuable for studying the consequences of enzyme loss on cholesterol biosynthesis and for validating CDPX2-related phenotypes.
Point Mutation
Point mutations identified in CDPX2 patients can be introduced into the endogenous EBP locus using CRISPR-Cas9 and homology-directed repair. Such models allow precise assessment of mutation effects on isomerase activity and sterol profiles.
Knock-in
Knock-in of epitope tags (e.g., FLAG, GFP) at the EBP locus enables studies of protein localization, interaction, and trafficking without overexpression artifacts. This approach is useful for understanding the enzyme's membrane topology.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression of EBP can be used to increase cholestenol delta-isomerase activity in cells. This is helpful for studying the effects of elevated enzyme levels on sterol metabolism and drug resistance.
How EDITGENE Supports cholestenol delta-isomerase activity Research
Researchers studying cholestenol delta-isomerase activity-related genes often need to determine whether a candidate gene is causally involved in cholesterol biosynthesis, disease phenotypes, or drug responses. EDITGENE provides a comprehensive suite of CRISPR-based services to create precisely engineered cell models for such investigations.
Contact EDITGENE today to design your custom CRISPR model for cholestenol delta-isomerase activity research.
Frequently Asked Questions About cholestenol delta-isomerase activity
What is cholestenol delta-isomerase activity?
Cholestenol delta-isomerase activity (GO:0047750) is the enzyme activity that catalyzes the conversion of 5-alpha-cholest-7-en-3-beta-ol to 5-alpha-cholest-8-en-3-beta-ol, a step in cholesterol biosynthesis.
What gene encodes cholestenol delta-isomerase activity?
The EBP gene encodes emopamil-binding protein, which exhibits cholestenol delta-isomerase activity.
What diseases are associated with cholestenol delta-isomerase activity?
Mutations in EBP cause X-linked dominant chondrodysplasia punctata 2 (CDPX2), and the enzyme is implicated in cancer and neuroprotection.
What is the reaction catalyzed by cholestenol delta-isomerase?
It isomerizes 5-alpha-cholest-7-en-3-beta-ol to 5-alpha-cholest-8-en-3-beta-ol.
Where is cholestenol delta-isomerase located in the cell?
The enzyme is localized to the endoplasmic reticulum membrane.
What are synonyms for cholestenol delta-isomerase activity?
Synonyms include cholestenol D-isomerase activity and delta7-cholestenol delta7-delta8-isomerase activity.
How can I study cholestenol delta-isomerase activity?
Enzymatic assays, structural biology, and CRISPR knockout models are common approaches.
Is cholestenol delta-isomerase activity conserved in yeast?
Yes, emopamil-binding protein exhibits delta8-delta7 sterol isomerase activity in yeast.
What drugs target cholestenol delta-isomerase?
Neuroprotective agents and 1-cyclohexyl-4-(4-arylcyclohexyl)piperazines bind to the enzyme.
What is the structural basis of cholestenol delta-isomerase activity?
The crystal structure of human sterol isomerase reveals a deep hydrophobic cavity for substrate binding and multidrug recognition.
Conclusion
Cholestenol delta-isomerase activity (GO:0047750) is a fundamental enzymatic function in cholesterol biosynthesis, catalyzed by emopamil-binding protein (EBP). Its role in human health is underscored by its association with CDPX2 and its potential as a drug target. Structural and functional studies have provided deep insights into its mechanism, and CRISPR-based models offer powerful tools for further research. Understanding this activity will continue to illuminate sterol metabolism and related diseases.
References
- 1. 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
- 2. 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
- 3. Long T et al.. 2019. Structural basis for human sterol isomerase in cholesterol biosynthesis and multidrug recognition.. Nat Commun 10(1):2452 PMID: 31165728
- 4. Silve S et al.. 1996. Emopamil-binding protein, a mammalian protein that binds a series of structurally diverse neuroprotective agents, exhibits delta8-delta7 sterol isomerase activity in yeast.. J Biol Chem 271(37):22434-40 PMID: 8798407
- 7. Yao H et al.. 2020. Thermostabilization of Membrane Proteins by Consensus Mutation: A Case Study for a Fungal Δ8-7 Sterol Isomerase.. J Mol Biol 432(18):5162-5183 PMID: 32105736
- 8. Hernandez O et al.. 2011. Gene expression during activation of Paracoccidioides brasiliensis conidia.. Yeast 28(11):771-81 PMID: 21960298
