Recombinant Bovine Lanosterol 14-alpha demethylase (CYP51A1)

What is Bovine Lanosterol 14-alpha demethylase (CYP51A1) and what is its functional role in mammalian systems?

Bovine Lanosterol 14-alpha demethylase (CYP51A1) is a cytochrome P450 enzyme that catalyzes a critical step in cholesterol biosynthesis. In mammals, it functions as a ubiquitously expressed membrane protein anchored to the endoplasmic reticulum . CYP51 specifically catalyzes the oxidative removal of the 14-alpha methyl group from lanosterol and 24,25-dihydrolanosterol (DHL), converting them to follicular fluid meiosis-activating sterol (FF-MAS) . This reaction requires molecular oxygen and NADPH as cofactors and involves three sequential oxidation steps.

The enzyme plays an essential role beyond direct cholesterol synthesis. CYP51 produces meiosis-activating sterols that were initially hypothesized to regulate meiosis and reproduction based on in vitro studies . The enzyme is highly conserved across species from fungi to mammals, indicating its evolutionary importance in sterol metabolism. In the sterol biosynthesis pathway, CYP51 functions downstream of lanosterol synthase and upstream of transmembrane 7 superfamily member 2 (TM7SF2), which converts FF-MAS to testicular meiosis-activating sterol (T-MAS) .

What structural modifications are necessary to create soluble recombinant bovine CYP51 for research applications?

Creating soluble recombinant bovine CYP51 requires specific structural modifications to overcome the inherent membrane-binding properties of the native enzyme. Researchers have successfully developed soluble monomeric forms through targeted modifications of the N-terminal membrane-spanning domain . These modifications typically involve:

• Truncation of the hydrophobic N-terminal membrane anchor

• Introduction of specific amino acid substitutions to enhance hydrophilicity

• Optimization of the remaining sequence to maintain proper protein folding

Two specific variants of bovine CYP51 (bCYP51) with different N-terminal truncations and modifications have been constructed and expressed in Escherichia coli at levels reaching 500 nmol/l . Comparative analysis revealed that the bCYP51-d1 variant exhibited approximately 10-fold better solubility than the bCYP51-d2 variant . Size-exclusion chromatography confirmed that bCYP51-d1 elutes as a single peak corresponding to its monomeric form, indicating minimal aggregation tendency .

Importantly, these structural modifications preserved enzymatic function. The activity of the soluble bCYP51-d1 variant remained comparable to that of recombinant human CYP51 with an intact membrane-spanning region . This combination of high solubility, monomeric behavior, and preserved catalytic activity makes the modified enzyme particularly valuable for crystallization attempts and detailed biochemical characterization.

How does the expression of CYP51 vary across different cell types and developmental stages?

The expression of CYP51 shows distinct patterns across cell types and developmental stages, reflecting its specialized functions in different contexts. In male reproductive tissues, stage-specific expression patterns of CYP51 during spermatogenesis have been documented . Detailed expression analyses revealed that CYP51 exhibits low expression levels in spermatogonia but increases significantly in primary spermatocytes and remains elevated in round and elongated spermatids .

This cell type-specific expression can be quantified using techniques such as quantitative RT-PCR with TaqMan assays targeting specific CYP51 exons. In studies of germ cell-specific CYP51 knockout mice, researchers separated germ cell populations through centrifugal elutriation to analyze expression in distinct cell types . The elutriation process yielded fractions containing primary spermatocytes (84% enriched), round spermatids (94% enriched), and elongated spermatids (97% enriched) .

Western blot analysis using antibodies against mammalian-specific CYP51 peptide sequences further confirms the protein-level expression patterns. In normal testes, CYP51 protein is readily detectable in whole tissue and in isolated germ cell fractions, particularly in round and elongated spermatids . This cell type-specific expression pattern suggests specialized roles for CYP51 during specific stages of germ cell development and differentiation.

What are the metabolic consequences of CYP51 inhibition or knockout in mammalian systems?

CYP51 inhibition or knockout produces specific and quantifiable metabolic alterations in the sterol biosynthesis pathway. Comprehensive metabolic profiling of male germ cell-specific CYP51 knockout mice revealed significant accumulation of CYP51 substrates coupled with depletion of downstream products . The following table summarizes the major metabolic changes observed:

Similar patterns were observed in isolated seminiferous tubules, with lanosterol increasing 2.5-fold and downstream products decreasing significantly (T-MAS by 65%, FF-MAS by 50%, desmosterol by 29%, and 7-DHC by 27%) .

These metabolic changes trigger compensatory responses in the cholesterol biosynthesis pathway. Gene expression analysis revealed a trend of upregulation for multiple cholesterogenic genes in knockout germ cells, particularly lanosterol synthase (Lss), which showed a statistically significant 1.3-fold upregulation in round spermatids . This compensatory response appears to be a cellular mechanism to maintain cholesterol homeostasis despite the CYP51 blockade.

How do the enzymatic properties of bovine CYP51 compare with CYP51 orthologs from other species?

Bovine CYP51 shares fundamental enzymatic mechanisms with CYP51 orthologs from other species but exhibits distinct properties that are important for comparative studies. All CYP51 family members catalyze the oxidative removal of the 14-alpha methyl group from sterol substrates, a reaction requiring molecular oxygen and electron transfer from NADPH via redox partner proteins.

Comparative analysis across species reveals significant evolutionary divergence. Fungi such as Aspergillus fumigatus possess two distinct CYP51 genes (cyp51A and cyp51B), each encoding functional 14-alpha sterol demethylase enzymes . These fungal enzymes share 40-70% amino acid sequence identity with each other and with CYP51 proteins from various species ranging from yeasts to filamentous fungi . In contrast, mammals typically express a single CYP51 gene, suggesting different evolutionary pressures on sterol metabolism across kingdoms.

The structural and functional differences between mammalian and fungal CYP51 enzymes have significant implications for inhibitor development. Azole antifungal drugs target fungal CYP51 with relatively higher selectivity compared to mammalian orthologs . This selectivity arises from structural differences in the enzyme active site, particularly in regions that interact with the azole nitrogen and side chains of inhibitor molecules.

These comparative differences make bovine CYP51 valuable for cross-species studies investigating evolutionary relationships between CYP51 enzymes and developing selective inhibitors that target fungal enzymes while sparing mammalian orthologs.

What is the relationship between CYP51 activity and meiosis-activating sterols in reproductive biology?

The relationship between CYP51 activity and meiosis-activating sterols (MASs) in reproductive biology is more complex than initially hypothesized. CYP51 produces FF-MAS, which is further converted to T-MAS by transmembrane 7 superfamily member 2 (TM7SF2) . These sterols were originally named "meiosis-activating" based on in vitro studies suggesting their role in regulating meiosis and oocyte maturation.

In vivo studies using male germ cell-specific CYP51 knockout mice have provided more nuanced insights into this relationship. Despite significant reduction in MAS levels (T-MAS decreased by 65-70% and FF-MAS by 33-50% in knockout mice), these animals maintained fertility and normal spermatogenesis . This unexpected finding challenges the hypothesis that MASs are essential for meiosis completion in male germ cells.

The metabolic profile changes in knockout mice were substantial and specific:

• Lanosterol increased 4.5-fold and 24,25-dihydrolanosterol increased 105-fold in whole testes

• FF-MAS decreased by 33% and T-MAS by 70% in whole testes

• Similar changes were observed in isolated seminiferous tubules

These findings suggest that while CYP51 and its sterol products may contribute to reproductive processes, there are likely redundant mechanisms or alternative pathways that can compensate for MAS reduction. The precise signaling roles of these sterols may be context-dependent or involve threshold effects rather than direct linear relationships with meiosis progression.

What are the optimal expression systems and conditions for producing active recombinant bovine CYP51?

The optimal expression system for recombinant bovine CYP51 is Escherichia coli, which has been successfully used to produce the enzyme at concentrations reaching 500 nmol/l . For maximum yield and activity, researchers should consider the following methodological details:

• Expression construct design:

  • Use modified versions with truncated N-terminal domains to improve solubility

  • Incorporate affinity tags (His-tag or similar) to facilitate purification

  • The bCYP51-d1 variant has demonstrated superior solubility and is recommended

• E. coli strain selection:

  • Choose strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3))

  • Consider strains with enhanced disulfide bond formation capability

  • Strains expressing rare tRNAs may improve translation efficiency

• Culture conditions:

  • Use rich media (such as Terrific Broth) for high cell density

  • Initial growth at 37°C to OD600 of 0.6-0.8

  • Reduce temperature to 20-25°C before induction

  • Add δ-aminolevulinic acid (0.5-1 mM) to enhance heme incorporation

  • Induce with low IPTG concentrations (0.1-0.5 mM)

  • Extended expression period (16-24 hours) at reduced temperature

• Harvest and lysis:

  • Gentle cell disruption methods (sonication or French press)

  • Include protease inhibitors to prevent degradation

  • Maintain reducing conditions to prevent oxidation of cysteine residues

The expressed protein variants should be purified and tested for solubility in the absence of detergent . Size-exclusion chromatography can confirm the monomeric state of the protein, which is important for subsequent enzymatic assays and structural studies. The bCYP51-d1 variant elutes as a single peak corresponding to its monomeric form, indicating minimal aggregation tendency .

What methods are most effective for measuring CYP51 enzymatic activity in vitro?

Multiple complementary methods can effectively measure CYP51 enzymatic activity in vitro, each with specific advantages for different research questions:

• LC-MS/MS-based sterol analysis:

  • Most specific and comprehensive approach

  • Simultaneously quantifies multiple sterols including substrates (lanosterol, DHL) and products (FF-MAS)

  • Requires sterol extraction followed by chromatographic separation

  • Can detect fold changes in metabolites (e.g., 4.5-fold increase in lanosterol, 105-fold increase in DHL in knockout studies)

• Spectrophotometric NADPH consumption assay:

  • Measures decrease in absorbance at 340 nm as NADPH is consumed

  • Allows continuous real-time monitoring of reaction progress

  • Less specific but useful for initial screening or kinetic studies

  • Requires correction for background NADPH oxidation

• Fluorescence-based assays:

  • Uses fluorogenic substrate analogs that change properties upon demethylation

  • Higher throughput than chromatographic methods

  • Less physiologically relevant than natural substrate assays

For a complete enzymatic reaction, the following components are essential:

• Purified recombinant CYP51 (typically 50-100 nM)

• NADPH-cytochrome P450 reductase (CPR) at 1:1 to 2:1 ratio to CYP51

• NADPH (typically 1 mM) or NADPH regenerating system

• Buffer system (typically 50-100 mM potassium phosphate, pH 7.4)

• Substrate (lanosterol or DHL, typically 25-50 μM)

• Appropriate detergent or lipid vesicles to solubilize substrate

Activity measurements should be performed under linear reaction conditions with respect to time and enzyme concentration. Results can be expressed as nmol product formed per minute per nmol CYP51, allowing direct comparison between different enzyme preparations and variants.

What techniques are available for isolating and analyzing CYP51 from different cell types for comparative studies?

Isolating and analyzing CYP51 from different cell types requires specialized techniques to preserve enzyme integrity and enable meaningful comparative studies. The following methodological approaches have been successfully employed:

• Cell type separation using centrifugal elutriation:

  • Allows separation of different germ cell populations based on size and density

  • Procedure involves resuspending cells in cold PBS and filtering through a 40 μm cell strainer

  • Loading the suspension into an elutriation chamber (e.g., JE-5.0) with rotor spinning at 2000 g

  • Incrementally increasing flow rate to collect different cell populations in separate fractions

  • Can achieve high enrichment (84% for primary spermatocytes, 94% for round spermatids, 97% for elongated spermatids)

• RNA extraction and quantitative RT-PCR:

  • TRIzol-based extraction provides high-quality RNA

  • TaqMan assays positioned in specific exons can quantify CYP51 expression

  • Primer design for bovine CYP51 should target conserved regions

  • β-Actin can serve as an internal control

  • Relative expression calculated using the standard ΔΔCt method

• Protein isolation and Western blot analysis:

  • Total proteins can be isolated using TRIzol Reagent

  • Protein pellet should be dissolved in 9 M urea, 2% CHAPS (1 hour, 60°C)

  • Sonication (5 × 1 min at 20 kHz with cooling intervals) improves solubilization

  • Bradford protein assay for quantification

  • SDS-PAGE loading approximately 80 μg protein per lane

  • Detection using antibodies against mammalian-specific CYP51 peptides

  • Visualization with ultra-sensitive enhanced chemiluminescent substrates

• Subcellular fractionation:

  • Differential centrifugation to separate endoplasmic reticulum membranes

  • Density gradient ultracentrifugation for higher purity fractions

  • Marker enzyme assays (e.g., NADPH-cytochrome c reductase) to confirm ER enrichment

These techniques enable researchers to compare CYP51 expression and characteristics across different cell types, developmental stages, or experimental conditions with high precision and reliability.

How should researchers design inhibitor screening assays for bovine CYP51?

Designing effective inhibitor screening assays for bovine CYP51 requires careful consideration of multiple factors to ensure reliability, specificity, and physiological relevance. A comprehensive approach should include:

• Primary screening assay design:

  • Select an appropriate detection method (NADPH consumption, product formation, or substrate depletion)

  • Optimize substrate concentration (typically at or slightly below Km)

  • Perform time-course studies to ensure linearity during the assay period

  • Include positive control inhibitors (e.g., ketoconazole or other azoles)

  • Screen at a fixed inhibitor concentration (typically 1-10 μM) initially

  • Include DMSO controls matching the highest solvent concentration used

• Secondary confirmation assays:

  • Dose-response studies for hit compounds (typically 8-10 concentrations)

  • IC50 determination using non-linear regression analysis

  • Counter-screens against related P450 enzymes to assess selectivity

  • Evaluation of potential interference with detection method

• Mechanism of inhibition studies:

  • Vary both substrate and inhibitor concentrations systematically

  • Perform global fitting to appropriate inhibition models

  • Determine inhibition constants (Ki) and inhibition mechanism

• Assay validation parameters:

• Considerations specific to recombinant bovine CYP51:

  • Use the well-characterized bCYP51-d1 variant for consistency

  • Include the complete electron transfer system (NADPH-P450 reductase)

  • Address substrate solubility issues with appropriate detergents or lipid systems

  • Consider potential effects of N-terminal modifications on inhibitor binding

This systematic approach enables identification of potent and selective CYP51 inhibitors while minimizing false positives and artifacts. The assay design should be tailored to the specific research objectives, whether developing research tools, comparative species studies, or potential therapeutic applications.

What controls and validation steps are essential when working with recombinant bovine CYP51?

Working with recombinant bovine CYP51 requires rigorous controls and validation steps to ensure reliable and reproducible results. The following comprehensive validation framework should be implemented:

• Protein quality validation:

  • Spectral characterization to confirm proper heme incorporation (CO-difference spectrum)

  • Size-exclusion chromatography to verify monomeric state and absence of aggregation

  • SDS-PAGE and Western blot analysis to confirm purity and identity

  • Mass spectrometry to verify primary sequence and detect potential modifications

• Functional validation:

  • Substrate binding titrations to determine binding affinity (Ks)

  • Enzymatic activity with natural substrates (lanosterol and DHL)

  • Comparison of catalytic parameters (Km, kcat) with literature values or native enzyme

  • Inhibition by known CYP51 inhibitors as positive controls

• Essential experimental controls:

  • No-enzyme controls to determine background reactions

  • No-substrate controls to measure endogenous activity

  • No-NADPH controls to distinguish P450-dependent from P450-independent reactions

  • Heat-inactivated enzyme controls to identify non-enzymatic effects

  • Vehicle controls (e.g., matching DMSO concentrations) for inhibitor studies

• Inter-assay validation:

  • Include internal standards across experiments

  • Regularly test reference inhibitors to ensure consistent response

  • Maintain detailed records of enzyme batch characteristics

  • Calculate and monitor coefficients of variation between assays

• System suitability criteria:

  • Minimum signal-to-noise ratio for activity measurements

  • Acceptable range for positive control inhibition

  • Criteria for linearity with respect to time and enzyme concentration

  • Maximum acceptable batch-to-batch variation

For structural studies, the bCYP51-d1 variant has shown promising characteristics, eluting as a single peak in size-exclusion chromatography and demonstrating limited tendency for non-specific oligomerization . These properties make it particularly suitable for crystallization attempts and consistent performance in functional assays.

How should researchers design experiments to study structure-function relationships in bovine CYP51?

Designing experiments to elucidate structure-function relationships in bovine CYP51 requires a multi-faceted approach combining protein engineering, functional characterization, and structural analysis. A comprehensive experimental strategy should include:

• Site-directed mutagenesis studies:

  • Target conserved residues identified through sequence alignment across species

  • Focus on putative substrate binding residues, heme coordination sites, and access channels

  • Create systematic alanine scanning mutations in regions of interest

  • Design mutations based on comparisons between bovine CYP51 and fungal orthologs

  • Use the soluble monomeric bCYP51-d1 variant as a template for mutagenesis

• Functional characterization of mutants:

  • Determine substrate binding affinity (Ks) through spectral titrations

  • Measure kinetic parameters (Km, kcat) with multiple substrates

  • Assess inhibitor binding and potency profiles

  • Evaluate protein stability through thermal denaturation assays

  • Compare activity against different sterol substrates to assess specificity changes

• Structural analysis approaches:

  • X-ray crystallography attempts with the highly soluble bCYP51-d1 variant

  • Hydrogen-deuterium exchange mass spectrometry to probe conformational dynamics

  • Molecular dynamics simulations to model substrate binding and protein motions

  • Homology modeling based on related CYP51 structures from other species

  • Small-angle X-ray scattering (SAXS) for solution-state structural information

• Correlation of structural and functional data:

• Domain swapping and chimeric constructs:

  • Exchange domains between bovine CYP51 and orthologs from other species

  • Create chimeras with fungal CYP51 to investigate species selectivity of inhibitors

  • Analyze the contribution of specific regions to substrate specificity

By systematically applying these approaches, researchers can develop a comprehensive understanding of how specific structural features of bovine CYP51 contribute to substrate recognition, catalytic efficiency, and inhibitor sensitivity. The high solubility and low tendency for non-specific oligomerization of bCYP51-d1 make it "a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme" .

How should metabolic profiling data for CYP51 function be analyzed and interpreted?

Metabolic profiling data for CYP51 function requires sophisticated analytical approaches to extract meaningful biological insights. Researchers should implement the following comprehensive strategy:

• Sterol quantification methodology:

  • Employ liquid chromatography-mass spectrometry (LC-MS) for precise identification and quantification

  • Include internal standards for each sterol class to ensure accurate quantification

  • Validate extraction efficiency for different sample types (whole tissue, cell fractions)

  • Ensure analytical methods can detect both substrate accumulation and product depletion

  • Develop methods sensitive enough to detect fold changes in key metabolites (e.g., 4.5-fold for lanosterol, 105-fold for DHL)

• Comparative analysis framework:

  • Calculate absolute concentrations of each sterol intermediate

  • Determine ratios between substrate and product (e.g., lanosterol/FF-MAS ratio) as indicators of enzymatic efficiency

  • Compare patterns across different experimental conditions and biological samples

  • Analyze both immediate CYP51 substrates and products, as well as downstream metabolites

• Statistical analysis approach:

  • Apply appropriate statistical tests based on data distribution

  • Account for multiple comparisons when analyzing many metabolites

  • Consider both statistical significance and magnitude of change

  • Perform correlation analysis between different sterols to identify coordinated changes

• Data visualization and interpretation:

  • Present comprehensive metabolic profiles in tabular format

  • Use pathway-based visualization to show relative changes in connected metabolites

  • Interpret changes in context of entire sterol biosynthesis pathway

  • Consider compensatory mechanisms (e.g., upregulation of other pathway enzymes)

• Integration with gene expression data:

  • Correlate sterol profiles with expression levels of CYP51 and other cholesterogenic genes

  • Identify potential regulatory relationships between metabolite levels and gene expression

  • Analyze feedback mechanisms that respond to altered sterol profiles

The metabolic consequences of CYP51 dysfunction should be interpreted as part of an integrated sterol homeostasis system. For example, the significant accumulation of lanosterol (4.5-fold) and DHL (105-fold) observed in CYP51 knockout studies was accompanied by compensatory upregulation of other cholesterogenic genes, suggesting coordinated pathway regulation .

What approaches are recommended for analyzing enzyme kinetic data for bovine CYP51?

Analyzing enzyme kinetic data for bovine CYP51 requires rigorous statistical and mathematical approaches to extract meaningful parameters and ensure reliability. Researchers should implement the following comprehensive strategy:

• Michaelis-Menten kinetics analysis:

  • Collect initial velocity data at multiple substrate concentrations (typically 6-8 concentrations spanning 0.2-5× Km)

  • Apply nonlinear regression using the Michaelis-Menten equation directly, rather than linearized plots

  • Calculate Km, Vmax, and catalytic efficiency (kcat/Km) with confidence intervals

  • Report enzyme concentration in active form (determined by CO difference spectroscopy)

  • Express activity in standard units (nmol product formed per min per nmol enzyme)

• Statistical validation of kinetic parameters:

  • Perform at least three independent experiments with different enzyme preparations

  • Calculate and report standard error or confidence intervals for all parameters

  • Apply goodness-of-fit tests (R², residual analysis) to validate model appropriateness

  • Consider alternative kinetic models if data shows systematic deviations from Michaelis-Menten

• Inhibition kinetics analysis:

  • Design experiments with multiple substrate and inhibitor concentrations

  • Apply global fitting to determine inhibition mechanism (competitive, non-competitive, mixed)

  • Calculate inhibition constants (Ki) with confidence intervals

  • Create Dixon or Cornish-Bowden plots to visualize inhibition patterns

  • Validate mechanism through statistical comparison of different models

• Data reporting standards:

• Comparative analysis considerations:

  • Standardize experimental conditions when comparing different enzyme variants

  • Use relative activity ratios when absolute values cannot be directly compared

  • Consider the impact of experimental variables (pH, temperature, ionic strength)

  • Account for potential differences in enzyme preparation when comparing results

These analytical approaches ensure robust determination of kinetic parameters that can be reliably used to characterize bovine CYP51 function, compare variants, and evaluate the effects of inhibitors or other modulators.

How can researchers effectively analyze protein-ligand interactions for bovine CYP51?

Analyzing protein-ligand interactions for bovine CYP51 requires integration of multiple biophysical and computational approaches to develop comprehensive binding models. Researchers should implement the following analytical framework:

• Spectral binding studies:

  • Perform type I and type II difference spectroscopy to characterize ligand interactions

  • Titrate ligand concentration to determine binding affinity (Ks)

  • Distinguish substrate-like (type I) from inhibitor-like (type II) spectral changes

  • Apply both equilibrium and kinetic binding approaches to determine on/off rates

  • Calculate and report standard errors and goodness-of-fit metrics

• Thermodynamic analysis:

  • Use isothermal titration calorimetry (ITC) to determine binding constants (Ka)

  • Determine thermodynamic parameters (ΔH, ΔS, ΔG) to characterize driving forces

  • Apply van't Hoff analysis by measuring binding at different temperatures

  • Interpret thermodynamic signature in relation to binding mechanism

• Structural data integration:

  • Leverage the properties of the soluble bCYP51-d1 variant for crystallographic studies

  • If crystal structures are available, analyze protein-ligand contacts and binding geometries

  • Apply molecular docking to predict binding modes for different ligands

  • Validate computational models through experimental testing of predicted mutations

• Structure-activity relationship analysis:

  • Systematically analyze binding data across structural analogs

  • Correlate structural features with binding affinity and spectral properties

  • Identify key pharmacophore elements required for high-affinity binding

  • Compare binding profiles between bovine CYP51 and orthologs from other species

• Data visualization and interpretation:

  • Present binding data in multiple formats (tables, binding isotherms, Scatchard plots)

  • Create molecular interaction diagrams highlighting key contacts

  • Compare observed binding patterns with known inhibitors of fungal CYP51

  • Interpret species-specific binding differences in structural context

The combined analysis of these complementary approaches provides a comprehensive understanding of how ligands interact with bovine CYP51. The availability of the soluble monomeric bCYP51-d1 variant, which exhibits "high solubility and low tendency to non-specific oligomer formation," facilitates these binding studies by providing a stable, well-behaved protein preparation .

What are the most significant challenges and future directions in bovine CYP51 research?

• Structural biology challenges:

  • Obtaining high-resolution crystal structures remains difficult despite improved solubility

  • Understanding dynamic conformational changes during the catalytic cycle

  • Elucidating structural basis for substrate recognition and regioselectivity

  • Mapping electron transfer interactions with redox partner proteins

• Functional characterization opportunities:

  • Developing more sensitive and specific activity assays for diverse substrates

  • Investigating potential roles of CYP51 beyond cholesterol biosynthesis

  • Exploring regulatory mechanisms controlling CYP51 activity in different tissues

  • Characterizing post-translational modifications affecting enzyme function

• Comparative biochemistry directions:

  • Expanding comparative studies between bovine CYP51 and orthologs from other species

  • Investigating the evolutionary divergence of CYP51 function across kingdoms

  • Understanding the structural basis for differences between mammalian and fungal enzymes

  • Leveraging these differences for selective inhibitor development

• Physiological role investigations:

  • Clarifying the precise functions of meiosis-activating sterols in reproductive biology

  • Investigating metabolic consequences of altered CYP51 function in different contexts

  • Exploring potential roles in signaling pathways beyond sterol metabolism

  • Understanding compensatory mechanisms that respond to CYP51 dysfunction