Recombinant Macaca fascicularis Lanosterol 14-alpha demethylase (CYP51A1)

What is Lanosterol 14-alpha demethylase (CYP51A1) and what are its primary functions?

CYP51A1 is an endoplasmic reticulum protein that participates in cholesterol synthesis by catalyzing the removal of the 14alpha-methyl group from lanosterol . It belongs to the cytochrome P450 superfamily of enzymes, which are monooxygenases that catalyze reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids . This enzyme possesses several biochemical functions including heme binding, iron ion binding, and sterol 14-demethylase activity . CYP51A1 is considered essential for development and growth, as mutations in cholesterol biosynthetic genes are often embryonic lethal .

How does Macaca fascicularis CYP51A1 compare to human CYP51A1?

Macaca fascicularis (cynomolgus monkey) CYP51A1 shares high sequence homology with human CYP51A1, making it a valuable model for studying human cholesterol metabolism. The Macaca fascicularis CYP51A1 protein consists of 503 amino acids and contains similar functional domains as the human ortholog . This includes conserved regions for heme binding, substrate recognition, and interaction with redox partners. The high degree of conservation reflects the enzyme's fundamental role in sterol metabolism across species and makes the cynomolgus monkey protein a suitable surrogate for human CYP51A1 in research applications.

What biochemical pathways involve CYP51A1?

CYP51A1 participates in several critical biochemical pathways:

What expression systems are commonly used for recombinant CYP51A1 production?

For recombinant CYP51A1 production, researchers typically use bacterial (E. coli), mammalian cells (including HEK293), or wheat germ cell-free systems . For the Macaca fascicularis variant specifically, expression has been successfully achieved using systems that maintain proper protein folding and heme incorporation. The choice of expression system depends on research objectives - bacterial systems offer high yield but may require refolding, while mammalian systems typically provide properly folded protein with correct post-translational modifications but at lower yields.

What purification strategies yield functional CYP51A1?

Functional CYP51A1 purification typically involves multi-step chromatography processes. Highly purified (>95% by SDS-PAGE) recombinant human CYP51A1 can be obtained through methods that preserve the native protein conformation . Common approaches include:

• Affinity chromatography using tags (His, GST, DDK, Myc, Flag, Avi, or Fc)

• Ion-exchange chromatography

• Size exclusion chromatography

The purified protein is typically stored in Tris-based buffer with 50% glycerol to maintain stability . For optimal results, purification should be conducted rapidly at 4°C to minimize protein degradation and maintain enzymatic activity.

How can researchers confirm proper folding and functionality of purified CYP51A1?

The gold standard for confirming proper folding of CYP51A1 is CO-difference spectroscopy, which should show a characteristic peak at approximately 450 nm for properly folded protein . A spectral peak at 420 nm indicates denatured protein (P420 form) . Figure 4 in one study demonstrates this technique, comparing wild-type CYP51-wt (black line), CYP51-R277L (red line), and CYP51-R431H (blue line) variants . Additional validation methods include substrate binding assays, enzyme activity measurements, and thermal stability assessments to ensure the purified protein is functionally active.

What CYP51A1 polymorphisms have been identified and what are their functional implications?

Several CYP51A1 polymorphisms have been identified with varying functional implications:

Molecular modeling studies suggest that variants like D152G and R277L affect enzymatic activity by reducing interaction with the obligatory redox partner POR (NADPH-cytochrome P450 oxidoreductase) .

How do mutations at key interaction sites affect CYP51A1 activity?

Mutations at key interaction sites can significantly impair CYP51A1 activity through several mechanisms:

• POR interaction sites: Variants like D152G and R277L show potentially lower binding to the obligatory redox partner POR, which would impair electron transfer necessary for catalysis .

• Substrate binding sites: Variants such as D152G and R431H demonstrate potentially lower affinity toward the substrate lanosterol, reducing catalytic efficiency .

• Azole interaction sites: Mutations at W245 and W250 to smaller serine residues could affect interaction with azole drugs, potentially altering individual susceptibility to azole treatments .

• Heme interaction sites: Y151 forms an H-bond with heme, and a change from tyrosine to aspartic acid (Y151D) is predicted to be damaging, as the distance to heme would become too large to form an H-bond .

Methodologically, researchers use molecular dynamic modeling, CO-difference spectroscopy, and in vitro enzyme assays to evaluate how these mutations affect protein function.

What clinical implications have been associated with CYP51A1 mutations?

CYP51A1 mutations have been associated with several clinical conditions:

• Pediatric cataracts: Mutation of Arg277 to Cys has been found in neurologically and systemically normal children with pediatric cataract .

• Severe congenital disorders: Three pathogenic variants (termination at Trp421, change of Ile312 to Thr, and Leu232 to Pro) cause not only cataract but also global developmental delay and hepatic failure .

• Cardiovascular parameters: Common CYP51A1 SNPs in noncoding regions have been associated with spontaneous premature labor, and lower LDL-C and TC in the second trimester of pregnancy .

• Metabolic factors: A common variant in the 3′UTR region has been associated with HbA1c and expression of genes in pancreas .

These findings suggest that including damaging CYP51A1 variants in genetic testing would benefit patients with conditions like pediatric cataract, neonatal hepatic failure, global developmental delay, and cardiovascular or metabolic diseases .

What are optimal conditions for CYP51A1 enzymatic activity assays?

Optimal conditions for CYP51A1 enzymatic assays typically include:

• Buffer system: Tris-based buffer systems are commonly used for maintaining stability

• pH range: Typically 7.0-7.5 for optimal activity

• Temperature: Usually 30-37°C for mammalian CYP51A1

• Cofactors: NADPH as electron donor and molecular oxygen

• Redox partners: Purified POR (NADPH-cytochrome P450 oxidoreductase) at appropriate ratios

• Substrate concentration: Lanosterol at concentrations near Km (often solubilized in detergent)

• Storage: 50% glycerol for long-term stability

Methods for activity detection include HPLC, LC-MS/MS, or spectrophotometric measurements of substrate depletion or product formation.

How should researchers approach the study of CYP51A1-POR interactions?

To study CYP51A1-POR interactions, researchers should consider:

• Reconstituted systems: Using purified components at varying ratios to determine optimal electron transfer

• Interaction mapping: Molecular modeling to identify key residues involved in the interaction

• Mutagenesis studies: Creating specific mutations (like D152G or R277L) to evaluate their effect on POR binding and electron transfer

• Spectroscopic methods: Monitoring changes in heme environment upon POR binding

• CO-difference spectroscopy: Evaluating the ability of mutants to form functional P450-CO complexes as seen in Figure 4 of the referenced study

When a variant fails to produce a P450 spectrum (as with R277L and R431H variants), this suggests critical impairment of either proper folding, heme incorporation, or POR interaction .

What strategies can be used to compare CYP51A1 across different species?

For cross-species comparison of CYP51A1, researchers should:

• Sequence alignment: Compare primary sequences to identify conserved and variable regions

• Homology modeling: Generate structural models based on available crystal structures

• Enzymatic characterization: Determine kinetic parameters (Km, Vmax, kcat) for each species variant

• Inhibition profiles: Compare susceptibility to inhibitors like azoles across species

• Expression systems: Express proteins from different species under identical conditions to minimize system-dependent variables

• Recombinant proteins: Use highly purified recombinant proteins like the Macaca fascicularis CYP51A1 described in the search results

• Chimeric proteins: Create chimeric enzymes to pinpoint regions responsible for species-specific differences

Such comparisons can reveal evolutionary adaptations and help predict species-specific drug interactions.

How can CYP51A1 research inform azole drug development?

CYP51A1 research provides valuable insights for azole drug development through:

• Structure-activity relationships: Analysis of how mutations at positions like Y151, W245, and W250 affect azole binding can guide the design of more potent or selective inhibitors .

• Species selectivity: Understanding differences between fungal and human CYP51A1 can help develop azoles with greater selectivity for fungal enzymes, reducing side effects.

• Resistance mechanisms: Studies of natural variants that affect azole binding (such as Y151D) help anticipate potential resistance mechanisms .

• Individual susceptibility: Research on human variants that alter azole interactions can inform personalized medicine approaches for antifungal therapy.

• Binding pocket analysis: Molecular modeling of the CYP51A1 active site with different azoles can reveal opportunities for novel drug design.

Methodologically, researchers should combine structural biology, biochemical assays, and clinical data to develop more effective and targeted azole drugs.

What techniques are most effective for studying CYP51A1 substrate specificity?

To effectively study CYP51A1 substrate specificity, researchers should employ:

• Enzyme kinetics: Determine Km and Vmax values for different substrates to quantify binding affinity and catalytic efficiency

• Spectral binding studies: Measure type I spectral shifts to quantify substrate binding in the absence of catalysis

• Product analysis: Use HPLC, GC-MS, or LC-MS/MS to identify and quantify reaction products

• Computational docking: Perform in silico docking studies to predict binding modes of various substrates

• Site-directed mutagenesis: Modify specific residues in substrate recognition sites to alter specificity

• Homology comparison: Compare substrate preferences across species to identify determinants of specificity

• Competitive binding assays: Use multiple substrates to assess preferential binding

These approaches together provide a comprehensive understanding of what structural features determine CYP51A1 substrate specificity.

How does CYP51A1 research contribute to understanding evolutionary conservation of P450 enzymes?

CYP51A1 research provides unique insights into P450 evolution because:

• Ancient origin: CYP51A1 is considered one of the oldest cytochrome P450 genes, found in all three eukaryotic phyla (fungi, plants, and animals) .

• Functional conservation: Despite sequence divergence, the catalytic mechanism removing the 14alpha-methyl group from lanosterol is preserved across diverse species.

• Sequence comparison: Analysis of Macaca fascicularis CYP51A1 alongside human and other mammalian orthologs reveals evolutionarily constrained regions .

• Natural polymorphisms: Study of natural variants highlights which protein regions can tolerate variation versus those under strict evolutionary pressure .

• Structural comparison: Conserved structural elements across species reveal fundamental requirements for P450 catalysis.

This research helps establish which features of P450 enzymes are indispensable for function and which can diversify to accommodate different physiological roles or environmental pressures.

What are common challenges in working with recombinant CYP51A1 and how can they be addressed?

Common challenges with recombinant CYP51A1 include:

• Improper folding: Some variants (like R277L and R431H) fail to produce characteristic P450 spectra . Solution: Optimize expression conditions, co-express with chaperones, or try different expression systems.

• Low expression yield: Solution: Optimize codon usage, expression temperature, and induction conditions. Consider using specialized expression systems like wheat germ or HEK293 cells .

• Poor heme incorporation: Solution: Supplement growth media with δ-aminolevulinic acid to enhance heme biosynthesis.

• Protein aggregation: Solution: Include glycerol (50%) in storage buffer , optimize protein concentration, and consider adding mild detergents.

• Insufficient purity: Solution: Implement multi-step purification strategies using combinations of affinity tags (His, GST, DDK, etc.) .

• Loss of activity during storage: Solution: Store at -20°C or -80°C, avoid repeated freeze-thaw cycles, and prepare working aliquots for short-term use at 4°C .

How can researchers interpret and troubleshoot abnormal CO-difference spectra?

When interpreting CO-difference spectra for CYP51A1:

• Normal spectrum: A properly folded P450 shows a characteristic peak at approximately 450 nm when complexed with CO, as demonstrated for wild-type CYP51A1 in Figure 4 of the referenced study .

• Abnormal 420 nm peak: This indicates conversion to the inactive P420 form, suggesting protein denaturation or improper heme coordination.

• Absent spectrum: As observed with R277L and R431H variants , this could indicate failure to incorporate heme, improper folding, or aggregation.

• Low amplitude: May indicate poor expression, heme incorporation issues, or partial denaturation.

Troubleshooting approaches include adjusting buffer conditions (pH, salt concentration), adding stabilizing agents, checking for proper reducing conditions, and testing different protein preparation methods.

What controls are essential when studying CYP51A1 variants?

Essential controls when studying CYP51A1 variants include:

• Wild-type enzyme: Always include wild-type CYP51A1 as a positive control for activity and spectral properties .

• Known inactive variants: Include previously characterized inactive variants (like those failing to produce P450 spectra) as reference points .

• Substrate-free controls: Necessary to establish baseline activity and spectral properties.

• System controls: When comparing across expression systems, include system-specific controls to account for background activities.

• Purification-matched samples: Ensure all variants undergo identical purification procedures to eliminate method-induced differences.

• Multiple protein batches: Test multiple independent protein preparations to ensure reproducibility and rule out batch-specific artifacts.

• Buffer controls: Include appropriate buffer controls, especially when testing inhibitors that may have limited solubility.

What are promising areas for future CYP51A1 research?

Promising future research directions for CYP51A1 include:

• Comprehensive variant analysis: Systematic characterization of all known natural variants to build a complete understanding of structure-function relationships .

• Personalized medicine applications: Including damaging CYP51A1 variants in genetic testing panels for conditions like pediatric cataract, neonatal hepatic failure, and metabolic diseases .

• Drug-enzyme interactions: Deeper investigation of how variants affect interaction with azoles and other drugs to guide personalized treatment approaches .

• Protein-protein interactions: Further characterization of CYP51A1 interactions with POR and other cellular components to understand the broader metabolic network .

• Evolutionary studies: Comparative analysis across species to understand how this ancient enzyme has been conserved while adapting to different physiological contexts .

• Structural biology: High-resolution structures of various CYP51A1 variants complexed with substrates and inhibitors to guide drug design.

• Tissue-specific regulation: Investigation of how CYP51A1 expression and activity are regulated in different tissues and pathological states.

How might CYP51A1 research impact clinical practice?

CYP51A1 research has several potential clinical implications:

• Genetic testing: Inclusion of damaging CYP51A1 variants in genetic testing panels for pediatric cataract, neonatal hepatic failure, global developmental delay, azole susceptibility, and cardiovascular and metabolic diseases .

• Pharmacogenomics: Identification of variants affecting azole drug metabolism could guide personalized dosing of antifungal medications .

• Disease biomarkers: CYP51A1 variants as potential biomarkers for susceptibility to metabolic disorders or drug responses.

• Novel therapeutic targets: Understanding of CYP51A1's role in various pathways could reveal new therapeutic approaches for metabolic disorders.

• Improved genetic counseling: Better understanding of genotype-phenotype relationships for CYP51A1 variants would enhance genetic counseling for affected families .

As research progresses, the clinical value of CYP51A1 as both a diagnostic marker and therapeutic target is likely to expand significantly.