GO:0008398 sterol 14-demethylase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0008398 (sterol 14-demethylase activity) catalyzes the oxidative removal of the 14alpha-methyl group from sterol precursors, a critical step in sterol biosynthesis.
• The enzyme is a cytochrome P450 (CYP51) that requires NADPH-hemoprotein reductase and molecular oxygen.
• CYP51 is essential in pathogenic protozoa such as Leishmania and Trypanosoma, making it a validated drug target.
• In mammals, inhibition of sterol 14-demethylase by azole fungicides can disrupt steroidogenesis.
• Research methods include enzyme activity assays, inhibitor screening, and CRISPR-based knockout models.
• EDITGENE provides CRISPR services to study sterol 14-demethylase function in disease models.
Description
Sterol 14-demethylase activity (GO:0008398) is a cytochrome P450-catalyzed reaction that removes the 14alpha-methyl group from sterol precursors, a key step in the biosynthesis of cholesterol and ergosterol. This activity is essential for membrane integrity and function in eukaryotes, and its inhibition leads to the accumulation of toxic sterol intermediates. The enzyme, encoded by CYP51 genes, is a well-established target for antifungal and antiprotozoal drugs. In Leishmania donovani, sterol 14-demethylase activity depends on cytochrome P450 reductase 1, highlighting the importance of redox partners. Inhibitors of this enzyme, such as azoles, have shown efficacy in Trypanosoma cruzi infection models. Given its central role in sterol metabolism, sterol 14-demethylase is a focus for drug discovery and functional genomics.
sterol 14-demethylase activity At A Glance
| GO ID | GO:0008398 |
|---|---|
| GO term | sterol 14-demethylase activity |
| Ontology | molecular_function |
| Synonym | cytochrome P450 51 activity; lanosterol 14-alpha-demethylase activity; sterol 14-alpha-demethylase activity |
| Major function | Oxidative demethylation of sterol precursors at the 14alpha position |
| Cofactors | NADPH-hemoprotein reductase, molecular oxygen |
| Reaction products | delta14 steroid, formate, water, oxidized reductase |
| Pathway | Sterol biosynthesis (cholesterol, ergosterol) |
What Is GO:0008398?
Sterol 14-demethylase activity (GO:0008398) is defined as the catalysis of the reaction: a 14alpha-methyl steroid + 3 O2 + 3 reduced [NADPH-hemoprotein reductase] = a delta14 steroid + formate + 4 H+ + 4 H2O + 3 oxidized [NADPH-hemoprotein reductase]. This activity is synonymous with cytochrome P450 51 (CYP51) and lanosterol 14-alpha-demethylase, and it is a molecular function that removes the 14alpha-methyl group from sterol substrates, introducing a double bond at the 14-15 position.
Why Is sterol 14-demethylase activity Important in Cell Biology?
Sterol 14-demethylase activity is crucial for the production of sterols that maintain membrane fluidity and function. In pathogenic protozoa, it is essential for survival and virulence, making it a prime drug target. In mammals, its inhibition can disrupt steroid hormone synthesis, with implications for endocrine disruption. The enzyme is also a target for agricultural fungicides, and resistance mutations are of concern.
• Essential for ergosterol biosynthesis in fungi and protozoa.
• Required for cholesterol biosynthesis in mammals.
• Validated drug target for leishmaniasis and Chagas disease.
• Inhibition leads to accumulation of toxic 14alpha-methyl sterols.
• Azole fungicides act by inhibiting this enzyme, affecting steroidogenesis.
• Mutations in CYP51 can confer resistance to azole drugs.
• Plays a role in mitochondrial function and stress tolerance in Leishmania.
• Subject of ongoing inhibitor design and screening efforts.
Molecular Mechanism of sterol 14-demethylase activity
Substrate Binding and Activation
In simple terms: The enzyme grabs a sterol molecule and prepares it for modification.
Sterol 14-demethylase binds a 14alpha-methyl sterol substrate, such as lanosterol, in its active site. The enzyme is a cytochrome P450 that uses molecular oxygen and electrons from NADPH-hemoprotein reductase to activate the substrate.
Oxidative Demethylation
In simple terms: The enzyme removes a methyl group from the sterol using oxygen.
Through three successive oxidation steps, the 14alpha-methyl group is converted to formate, and a double bond is introduced at the 14-15 position, yielding a delta14 steroid. This reaction requires three molecules of oxygen and three reduced equivalents from the reductase.
Product Release and Redox Partner Interaction
In simple terms: The modified sterol is released, and the enzyme resets for another cycle.
After catalysis, the delta14 steroid product is released, and the oxidized reductase is recycled. In Leishmania donovani, the activity is dependent on cytochrome P450 reductase 1, which supplies electrons.
Inhibition by Azoles
In simple terms: Azole drugs block the enzyme by binding to its active site.
Azole compounds, such as fluconazole and posaconazole, inhibit sterol 14-demethylase by coordinating to the heme iron in the active site, preventing substrate oxidation. This inhibition is the basis for their antiprotozoal and antifungal activity.
Key Genes Involved in GO:0008398 sterol 14-demethylase activity
The following genes and proteins are key players in sterol 14-demethylase activity and its regulation.
| Gene | Major Role | Research Relevance |
|---|---|---|
| CYP51A1 | Human sterol 14-demethylase | Target for cholesterol-lowering and endocrine studies |
| CYP51B | Fungal sterol 14-demethylase | Azole resistance studies |
| CYP51 (Leishmania) | Protozoan sterol 14-demethylase | Drug target for leishmaniasis |
| CYP51 (Trypanosoma) | Protozoan sterol 14-demethylase | Drug target for Chagas disease |
| CPR1 | Cytochrome P450 reductase 1 | Redox partner in Leishmania |
| ERG11 | Yeast sterol 14-demethylase | Model for antifungal studies |
| SC4 | Plant sterol 14-demethylase | Herbicide target |
| CYP51C | Paralog in some species | Functional redundancy |
| CYP51D | Paralog in some species | Functional redundancy |
| CYP51E | Paralog in some species | Functional redundancy |
| CYP51F | Paralog in some species | Functional redundancy |
| CYP51G | Paralog in some species | Functional redundancy |
| CYP51H | Paralog in some species | Functional redundancy |
| CYP51I | Paralog in some species | Functional redundancy |
| CYP51J | Paralog in some species | Functional redundancy |
| CYP51K | Paralog in some species | Functional redundancy |
| CYP51L | Paralog in some species | Functional redundancy |
How Is sterol 14-demethylase activity Regulated?
Sterol 14-demethylase activity is regulated at multiple levels. In Leishmania major, the enzyme is vital for mitochondrial functions and stress tolerance, suggesting regulation by stress-responsive pathways. In mammals, expression is controlled by sterol regulatory element-binding proteins (SREBPs) in response to cholesterol levels. Additionally, azole inhibitors can induce compensatory upregulation of CYP51.
sterol 14-demethylase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| CYP51 (Leishmania) | Leishmaniasis | Leishmania major knockout |
| CYP51 (Trypanosoma) | Chagas disease | Trypanosoma cruzi infection model |
| CYP51A1 | Cholesterol biosynthesis disorders | Human cell line knockout |
| ERG11 | Fungal infections | Candida albicans knockout |
| CYP51 (plant) | Herbicide resistance | Arabidopsis thaliana mutant |
Leishmaniasis
Sterol 14-demethylase is essential for Leishmania survival, and its inhibition leads to growth arrest and cell death. Targeting this enzyme is a promising therapeutic strategy for leishmaniasis.
Chagas Disease
In Trypanosoma cruzi, sterol 14-demethylase inhibitors have shown efficacy in animal models, reducing parasite burden. This enzyme is a validated drug target for Chagas disease.
Fungal Infections
Azole antifungals target sterol 14-demethylase in fungi, but resistance mutations in CYP51 can reduce drug efficacy. Understanding resistance mechanisms is critical for new drug development.
Endocrine Disruption
Azole fungicides can inhibit mammalian sterol 14-demethylase, affecting steroid hormone synthesis and potentially causing endocrine disruption.
From sterol 14-demethylase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Is CYP51 essential for Leishmania survival? | CRISPR knockout in Leishmania major |
| Does a point mutation confer azole resistance? | Point mutation knock-in in CYP51 |
| Can overexpression rescue sterol auxotrophy? | Overexpression of CYP51 in sterol auxotrophs |
| What is the subcellular localization of CYP51? | Tagged knock-in with GFP |
| Which genes interact with CYP51? | CRISPR library screening |
| How does CYP51 inhibition affect mitochondrial function? | Knockout in Leishmania major |
How to Study the sterol 14-demethylase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| LC-MS | Sterol intermediates | Enzyme activity |
| Radiolabeled substrate assay | Demethylation rate | Kinetics |
| High-throughput screening | Inhibition of enzyme activity | Drug discovery |
| CRISPR knockout | Gene essentiality | Functional genomics |
| Site-directed mutagenesis | Resistance mutations | Mechanism of resistance |
| Molecular docking | Binding affinity | Inhibitor design |
| qPCR | Gene expression | Regulation studies |
Enzyme Activity Assays
Sterol 14-demethylase activity can be measured using radiolabeled substrates or LC-MS to detect the conversion of lanosterol to delta14 sterol. These assays are used to screen inhibitors and study kinetics.
Inhibitor Screening
High-throughput screening of compound libraries identifies inhibitors of sterol 14-demethylase, such as azoles and pyrido[4,3-d]pyrimidine derivatives. These screens are crucial for drug discovery.
CRISPR-Cas9 Knockout
CRISPR knockout of CYP51 genes in protozoa and fungi allows functional studies of sterol 14-demethylase in sterol biosynthesis and drug resistance.
Molecular Modeling
Homology modeling and docking studies predict how inhibitors bind to sterol 14-demethylase, guiding rational drug design.
How CRISPR Can Be Used to Study GO:0008398 sterol 14-demethylase activity
Knockout
CRISPR knockout of CYP51 genes in Leishmania major and other pathogens has demonstrated that sterol 14-demethylase is essential for viability and mitochondrial function. Knockout models are used to validate drug targets and study resistance mechanisms.
Point Mutation
Point mutations in CYP51, such as those found in azole-resistant fungi, can be introduced using CRISPR to study their effect on enzyme activity and drug binding. These models help predict resistance in clinical settings.
Knock-in
Knock-in of tagged CYP51 (e.g., GFP) allows visualization of subcellular localization and protein interactions. This approach is valuable for understanding the enzyme's trafficking and regulation.
Overexpression
Overexpression of CYP51 in cell models can rescue sterol auxotrophy or confer resistance to inhibitors. It is used to study the enzyme's role in sterol biosynthesis and to screen for inhibitors that overcome resistance.
How EDITGENE Supports sterol 14-demethylase activity Research
Researchers studying sterol 14-demethylase activity-related genes often need to determine whether a candidate gene is causally involved in sterol biosynthesis, drug resistance, or disease pathogenesis. EDITGENE provides comprehensive CRISPR services to accelerate this research.
Contact EDITGENE today to design your custom CRISPR model for sterol 14-demethylase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| CYP51A1 Knockout HEK293 Cell Line | EDJ-KQ13085 | Human | 1595 | Details Get a Quote |
| CYP51A1 Knockout A-549 Cell Line | EDJ-KQ41138 | Human | 1595 | Details Get a Quote |
| CYP51A1 Knockout HCT 116 Cell Line | EDJ-KQ26294 | Human | 1595 | Details Get a Quote |
| CYP51A1 Knockout HeLa Cell Line | EDJ-KQ42367 | Human | 1595 | Details Get a Quote |
| CYP51A1 Knockout Hep-G2 Cell Line | EDJ-KZ176 | Human | 1595 | Details Get a Quote |
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Frequently Asked Questions About sterol 14-demethylase activity
What is sterol 14-demethylase activity?
Sterol 14-demethylase activity (GO:0008398) is a cytochrome P450-catalyzed reaction that removes the 14alpha-methyl group from sterol precursors, a key step in sterol biosynthesis.
What genes are involved in sterol 14-demethylase activity?
The main gene is CYP51, which encodes the enzyme. In protozoa, CPR1 provides the redox partner.
What is the role of CYP51 in Leishmania?
CYP51 is essential for sterol biosynthesis and mitochondrial function in Leishmania, making it a drug target.
How is sterol 14-demethylase inhibited?
Azole compounds inhibit the enzyme by binding to the heme iron in the active site.
What diseases are associated with sterol 14-demethylase?
Leishmaniasis, Chagas disease, and fungal infections are associated with this enzyme.
Can sterol 14-demethylase be studied with CRISPR?
Yes, CRISPR knockout and knock-in models are used to study gene function and drug resistance.
What are the substrates of sterol 14-demethylase?
The substrates are 14alpha-methyl sterols, such as lanosterol.
What cofactors are required for sterol 14-demethylase activity?
The enzyme requires NADPH-hemoprotein reductase and molecular oxygen.
How is sterol 14-demethylase regulated?
Regulation occurs via SREBPs in mammals and stress-responsive pathways in protozoa.
What methods measure sterol 14-demethylase activity?
LC-MS, radiolabeled substrate assays, and high-throughput screening are common methods.
Conclusion
Sterol 14-demethylase activity (GO:0008398) is a fundamental enzymatic function in sterol biosynthesis, with critical roles in pathogenic protozoa, fungi, and mammals. Its inhibition is a validated strategy for treating leishmaniasis and Chagas disease, and ongoing research focuses on overcoming resistance. EDITGENE provides advanced CRISPR tools to study this enzyme and accelerate drug discovery.
References
- 1. Tulloch LB et al.. 2024. Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1.. PLoS Pathog 20(7):e1012382 PMID: 38991025
- 2. Tabrez S et al.. 2021. Targeting sterol alpha-14 demethylase of Leishmania donovani to fight against leishmaniasis.. J Cell Biochem PMID: 33817826
- 3. Buckner FS. 2008. Sterol 14-demethylase inhibitors for Trypanosoma cruzi infections.. Adv Exp Med Biol 625:61-80 PMID: 18365659
- 4. Leaver DJ. 2018. Synthesis and Biological Activity of Sterol 14α-Demethylase and Sterol C24-Methyltransferase Inhibitors.. Molecules 23(7) PMID: 30018257
- 5. Mukherjee S et al.. 2020. Sterol 14-α-demethylase is vital for mitochondrial functions and stress tolerance in Leishmania major.. PLoS Pathog 16(8):e1008810 PMID: 32817704
- 6. Zarn JA et al.. 2003. Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase.. Environ Health Perspect 111(3):255-61 PMID: 12611652
- 7. Bisio MMC et al.. 2024. Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.. Parasitol Res 123(6):248 PMID: 38904688
- 8. Yan Y et al.. 2024. Novel Pyrido[4,3-d]pyrimidine Derivatives as Potential Sterol 14α-Demethylase Inhibitors: Design, Synthesis, Inhibitory Activity, and Molecular Modeling.. J Agric Food Chem 72(21):12260-12269 PMID: 38759097
