Recombinant Bovine Squalene synthase (FDFT1)

What is the biochemical function of Bovine Squalene Synthase (FDFT1) in cellular metabolism?

Farnesyl-Diphosphate Farnesyltransferase 1 (FDFT1), commonly known as Squalene Synthase, is a membrane-associated enzyme occupying a critical branch point in the mevalonate pathway . This bifunctional enzyme catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP), followed by the NADPH-dependent rearrangement and reduction of PSPP to produce squalene . This two-step reaction represents the first pathway-specific step in sterol biosynthesis in eukaryotes and hopane biosynthesis in certain bacteria .

To investigate FDFT1's metabolic function experimentally, researchers should consider:

• Employing isotope labeling with 13C-acetate to trace carbon flow through the pathway

• Conducting metabolomic profiling before and after FDFT1 inhibition

• Analyzing downstream sterol intermediates using LC-MS or GC-MS techniques

• Examining gene expression changes in response to FDFT1 modulation using RT-qPCR

The position of FDFT1 at a key branch point makes it particularly significant for understanding cholesterol homeostasis and potential therapeutic interventions targeting sterol metabolism.

How is FDFT1 conserved across species and what implications does this have for research models?

FDFT1 demonstrates remarkable evolutionary conservation, reflecting its fundamental role in sterol/isoprenoid biosynthesis across diverse organisms. Comparative analysis reveals:

• The enzyme's catalytic core and binding sites for farnesyl diphosphate are highly conserved from bacteria to mammals

• FDFT1 orthologs exist in bacteria (where they function in hopane biosynthesis) and in eukaryotes (where they direct sterol biosynthesis)

• Zebrafish (Danio rerio) fdft1 is predicted to have functions similar to mammalian FDFT1, including cholesterol biosynthesis and farnesyl diphosphate metabolism

When selecting research models, this conservation pattern suggests that:

• Bacterial FDFT1 homologs (such as from T. elongatus) may serve as simplified models for studying basic catalytic mechanisms

• Zebrafish models can provide insights into vertebrate FDFT1 function in development

• Bovine FDFT1 shares sufficient homology with human FDFT1 to serve as a relevant model for human health applications

Researchers should employ phylogenetic analysis and sequence alignment tools (BLAST, Clustal Omega) to identify conserved motifs across species before selecting appropriate experimental models. The high conservation of FDFT1 suggests that findings from bovine studies may have translational relevance to human health research.

What role does FDFT1 play in cancer biology and how might this inform therapeutic strategies?

FDFT1 has emerged as a significant player in cancer biology through multiple mechanisms related to cellular metabolism and signaling. Recent research indicates that FDFT1 contributes to metabolic reprogramming, cell proliferation, and invasion in various tumor types . The mechanistic connections between FDFT1 and cancer include:

• Altered cholesterol homeostasis affecting membrane fluidity and lipid raft formation

• Disruption of isoprenoid availability for protein prenylation (affecting Ras/Rho GTPase signaling)

• Metabolic adaptations supporting rapid cancer cell proliferation

• Potential interactions with oncogenic signaling pathways

To investigate FDFT1's role in cancer, researchers should implement a multi-faceted approach:

• Expression analysis:

  • Compare FDFT1 mRNA and protein levels between tumor and matched normal tissues

  • Correlate expression with clinical parameters (stage, grade, prognosis)

  • Use tissue microarrays for high-throughput immunohistochemical analysis

• Functional studies:

  • Generate stable FDFT1 knockdown and overexpression cell lines

  • Assess changes in proliferation, migration, invasion, and apoptosis

  • Examine metabolomic alterations using mass spectrometry approaches

• Therapeutic exploration:

  • Test FDFT1 inhibitors alone and in combination with established cancer therapies

  • Investigate synthetic lethality with other metabolic pathway inhibitors

  • Develop cancer cell-specific targeting strategies to minimize off-target effects

This research direction may ultimately position FDFT1 as both a prognostic biomarker and therapeutic target for specific cancer types .

How do post-translational modifications affect Bovine FDFT1 activity and what challenges does this present for recombinant protein studies?

Post-translational modifications (PTMs) significantly impact FDFT1 activity, creating important distinctions between native and recombinant forms of the enzyme. The major PTMs affecting Bovine FDFT1 include:

• Phosphorylation of serine/threonine residues that can modulate enzymatic activity

• Glycosylation patterns affecting protein stability and membrane association

• Prenylation of C-terminal residues influencing membrane localization

• Disulfide bond formation affecting tertiary structure and catalytic activity

These modifications present several methodological challenges for researchers working with recombinant Bovine FDFT1:

• Expression system limitations:

  • E. coli lacks machinery for mammalian-type glycosylation and most PTMs

  • Recombinant FDFT1 from bacterial systems may exhibit altered kinetic parameters

• Activity discrepancies:

  • Native FDFT1 typically shows higher specific activity than recombinant versions

  • Substrate affinity (Km) values may differ due to subtle conformational changes

• Membrane association differences:

  • Recombinant FDFT1 often lacks proper membrane insertion observed in native enzyme

  • Solubilization strategies must be carefully optimized to maintain activity

To address these challenges, researchers should:

• Consider eukaryotic expression systems (insect cells, yeast) for more authentic PTMs

• Perform comprehensive kinetic characterization comparing native and recombinant forms

• Implement activity normalization protocols when comparing different preparations

• Use detergent screening to identify optimal solubilization conditions

The absence of proper PTMs in recombinant preparations should always be acknowledged as a potential limitation when interpreting experimental results.

What are the catalytic mechanisms of Bovine FDFT1 and how can they be experimentally verified?

Bovine FDFT1 employs a complex two-step catalytic mechanism to convert farnesyl diphosphate to squalene. The reaction proceeds through:

• Condensation phase:

  • Two farnesyl diphosphate (FPP) molecules bind in the active site

  • Mg²⁺ ions coordinate with conserved aspartate residues to position substrates

  • Ionization of one FPP generates a carbocation that attacks the second FPP

  • Formation of a cyclopropylcarbinyl diphosphate intermediate

  • Rearrangement to presqualene diphosphate (PSPP)

• Reduction/rearrangement phase:

  • NADPH-dependent reduction of PSPP

  • Skeletal rearrangement with bond breaking/formation

  • Release of squalene as the final product

To experimentally verify these mechanisms, researchers should employ:

• Site-directed mutagenesis targeting:

  • Conserved DXXXD motifs (Mg²⁺ coordination)

  • Tyrosine and phenylalanine residues (carbocation stabilization)

  • Residues involved in NADPH binding

• Reaction intermediate trapping:

  • Use of non-hydrolyzable FPP analogs

  • Low-temperature reaction conditions to slow catalysis

  • Rapid quenching techniques followed by LC-MS analysis

• Kinetic isotope effect studies:

  • Deuterium-labeled substrates to identify rate-limiting steps

  • Position-specific labeling to track hydrogen transfers

• Spectroscopic approaches:

  • Pre-steady-state kinetics using stopped-flow techniques

  • Fluorescence resonance energy transfer (FRET) to monitor conformational changes

These experimental strategies provide complementary data to build a comprehensive understanding of FDFT1's catalytic mechanism, which is essential for rational inhibitor design and enzyme engineering applications.

What are the optimal conditions for expressing soluble recombinant Bovine FDFT1 in E. coli?

Expressing soluble recombinant Bovine FDFT1 in E. coli presents significant challenges due to its membrane-associated nature. Based on empirical findings from related FDFT1 proteins, researchers should implement the following optimized protocol:

Critical factors for success include:

• Addition of glycerol to all buffers, which significantly enhances FDFT1 solubility as demonstrated in related SQS proteins

• Careful temperature management during cell disruption to prevent aggregation

• Immediate clarification of lysate by high-speed centrifugation (20,000 × g for 30 minutes)

• Addition of protease inhibitors to prevent degradation during purification

When optimized, this approach can yield 1-3 mg of soluble recombinant Bovine FDFT1 per liter of culture, sufficient for most biochemical and structural studies.

What purification strategies yield the highest purity and activity for recombinant Bovine FDFT1?

Purification of recombinant Bovine FDFT1 to high homogeneity while preserving enzymatic activity requires a multi-step chromatographic approach. The following purification strategy has been empirically optimized:

• Initial capture by Immobilized Metal Affinity Chromatography (IMAC):

  • Equilibrate Ni-NTA resin with 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, 10 mM imidazole

  • Bind clarified lysate containing His-tagged FDFT1

  • Wash extensively with increasing imidazole concentrations (20-40 mM)

  • Elute with 250-300 mM imidazole in the same buffer

  • Expected purity at this stage: 60-70%

• Intermediate purification by Ion Exchange Chromatography:

  • Dialyze IMAC elution against 20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10% glycerol

  • Apply to Q-Sepharose column

  • Elute with linear NaCl gradient (50-500 mM)

  • Pool fractions containing FDFT1 based on SDS-PAGE analysis

  • Expected purity after this step: 80-85%

• Polishing by Size Exclusion Chromatography:

  • Apply concentrated protein to Superdex 200 column

  • Elute with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol

  • Collect fractions corresponding to monomeric FDFT1

  • Final purity should exceed 90%

Critical factors for maintaining activity:

• Inclusion of 10% glycerol in all buffers to stabilize the enzyme

• Addition of 1 mM DTT to prevent oxidation of critical cysteine residues

• Maintaining temperature at 4°C throughout purification

• Avoiding freeze-thaw cycles by preparing single-use aliquots

For storage, the purified enzyme should be supplemented with additional glycerol (final concentration 20%) and stored at -80°C. For extended storage, addition of carrier protein (0.1% BSA) is recommended to prevent activity loss .

What enzyme assay methods can accurately measure Bovine FDFT1 activity?

Accurately measuring Bovine FDFT1 activity requires careful consideration of its bifunctional nature and membrane association. The following assay methodologies provide complementary approaches for comprehensive enzyme characterization:

• Radioisotope-based endpoint assays:

  • Substrate: [14C]-labeled farnesyl diphosphate (FPP)

  • Reaction conditions: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM NADPH, 50 μM FPP, 37°C

  • Detection: Organic extraction followed by thin-layer chromatography or HPLC

  • Quantification: Scintillation counting of isolated squalene product

  • Sensitivity: Can detect pmol quantities of product

  • Advantages: Direct quantification of final product; high sensitivity

• Spectrophotometric continuous assays:

  • Principle: Monitor NADPH oxidation at 340 nm (ε = 6220 M⁻¹cm⁻¹)

  • Reaction mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM NADPH, 50-100 μM FPP

  • Control: Background NADPH oxidation must be subtracted

  • Calculation: 1 mole NADPH consumed per mole squalene formed

  • Advantages: Real-time monitoring; suitable for kinetic studies

• HPLC-based product quantification:

  • Reaction termination: Methanol precipitation

  • Extraction: Hexane or equivalent organic solvent

  • Separation: Reverse-phase HPLC with C18 column

  • Detection: UV absorbance at 210 nm or evaporative light scattering

  • Standard curve: Pure squalene standards (0.1-100 μM)

  • Advantages: No radioactivity; direct product quantification

• Coupled enzyme assay for first-step activity:

  • Principle: Measure presqualene diphosphate (PSPP) formation

  • Detection: Malachite green assay for released pyrophosphate

  • Advantage: Isolates first catalytic step from second step

For all assays, the following controls are essential:

• Heat-inactivated enzyme (negative control)

• Known FDFT1 inhibitor (e.g., zaragozic acid) as positive control for inhibition

• Time-course measurements to ensure linearity during the rate determination period

• Protein concentration determination by multiple methods to enable specific activity calculation

Specific activity should be reported as nmol product formed per minute per mg protein under standard conditions (37°C, pH 7.5).

How can researchers address the challenge of membrane protein solubility when working with FDFT1?

The membrane-associated nature of Bovine FDFT1 presents significant solubility challenges for researchers. Based on empirical findings from studies with related proteins, the following methodological strategies effectively address these challenges:

• Detergent-based solubilization approach:

  • Systematic screening of detergent classes using a tiered approach:

    • Mild non-ionic detergents: n-dodecyl-β-D-maltoside (DDM), Triton X-100

    • Zwitterionic detergents: CHAPS, Fos-Choline

    • Bile salt derivatives: sodium cholate, CHAPSO

  • Optimal detergent concentration: typically 2-5× critical micelle concentration (CMC)

  • Assessment method: Centrifugation followed by SDS-PAGE and activity assays of supernatant

• Buffer optimization strategies:

  • Addition of glycerol (10-20%) significantly enhances FDFT1 solubility

  • Inclusion of mild solubilizers (0.4M urea) helps maintain solubility without denaturation

  • Buffer pH optimization (typically 7.5-8.0 for maximal stability)

  • Addition of stabilizing osmolytes (sucrose, trehalose) at 5-10%

• Protein engineering approaches:

  • Truncation of C-terminal membrane-spanning domain

  • Fusion with solubility-enhancing partners (MBP, SUMO, TrxA)

  • Introduction of surface mutations to reduce hydrophobic patches

• Advanced solubilization technologies:

  • Nanodisc incorporation for functional studies

  • Amphipol stabilization after initial detergent solubilization

  • Styrene maleic acid lipid particles (SMALPs) for native-like membrane environment

When working with inclusion bodies:

• Solubilize with 6-8M urea or 6M guanidine hydrochloride

• Implement stepwise dialysis with decreasing denaturant concentrations

• Add glycerol, mild detergents, and L-arginine during refolding

• Monitor secondary structure recovery using circular dichroism

The empirical finding that glycerol significantly enhances FDFT1 solubility across species boundaries suggests this should be the first-line approach before attempting more complex solubilization strategies .

What methods can be used to assess enzyme kinetics for the bifunctional reactions of FDFT1?

Analyzing the complex kinetics of Bovine FDFT1 requires specialized methodologies that account for its bifunctional nature. Researchers should implement the following approaches:

Example experimental design for determining kinetic parameters:

This comprehensive kinetic analysis provides insights into the catalytic mechanism and can inform inhibitor design targeting either the first or second catalytic step independently.

How can researchers effectively compare data from different FDFT1 orthologues in evolutionary studies?

Comparative analysis of FDFT1 orthologs requires careful methodological considerations to ensure valid cross-species comparisons. Researchers should implement the following strategies:

• Sequence-based comparative methods:

  • Multiple sequence alignment using MUSCLE or Clustal Omega algorithms

  • Calculation of sequence identity and similarity percentages

  • Identification of conserved catalytic motifs across species

  • Phylogenetic tree construction using maximum likelihood methods

  • Selection pressure analysis (dN/dS ratios) to identify functionally important regions

• Structural comparison approaches:

  • Homology modeling of different orthologs based on available crystal structures

  • Superimposition of structural models to identify conserved spatial arrangements

  • Active site architecture analysis focused on substrate binding residues

  • Molecular dynamics simulations to compare dynamic properties

  • Quantitative metrics: RMSD values for backbone atoms, conservation of hydrogen bonding networks

• Functional parameter normalization:

  • Standardized expression and purification protocols across orthologs

  • Identical assay conditions for kinetic measurements

  • Calculation of catalytic efficiency (kcat/Km) for meaningful comparisons

  • Temperature adaptation analysis using Arrhenius plots

  • pH profile comparison with normalized maximum activity

• Statistical methods for evolutionary interpretation:

  • Ancestral sequence reconstruction to infer evolutionary trajectories

  • Correlation analysis between sequence/structural features and functional parameters

  • Principal component analysis to identify major sources of variation

  • Hierarchical clustering to group functionally similar orthologs

When comparing bovine FDFT1 with other mammalian orthologs (e.g., human, mouse), researchers should consider the high degree of conservation in the catalytic core while recognizing potential differences in membrane association domains and regulatory regions. For comparisons with more distant orthologs (e.g., fish or bacterial SQS), focus on the conserved catalytic mechanism while acknowledging the significant evolutionary distance .

This comprehensive approach enables meaningful evolutionary insights while avoiding oversimplified comparisons based solely on sequence identity.

What are the potential therapeutic applications of FDFT1 inhibitors beyond cholesterol reduction?

FDFT1 inhibition offers therapeutic potential beyond traditional cholesterol management, with several promising research directions emerging:

• Cancer treatment strategies:

  • FDFT1 inhibition disrupts metabolic reprogramming in cancer cells

  • Synergistic effects observed when combined with conventional chemotherapies

  • Selective toxicity toward rapidly proliferating cells with high cholesterol demands

  • Potential to overcome drug resistance mechanisms in certain tumor types

• Anti-parasitic applications:

  • Parasites (Trypanosoma, Leishmania) rely on sterol biosynthesis for survival

  • Structural differences between host and parasite FDFT1 enable selective targeting

  • Lower toxicity compared to current anti-parasitic medications

  • Potential for combination therapy with existing antiparasitic drugs

• Neurodegenerative disease modification:

  • Aberrant cholesterol metabolism implicated in Alzheimer's pathology

  • FDFT1 inhibition may reduce amyloid-β production through membrane effects

  • Potential neuroprotective effects by modulating neuroinflammation

  • Brain-penetrant FDFT1 inhibitors show promise in preliminary models

• Immunomodulatory effects:

  • Altered lipid raft composition affects T-cell receptor signaling

  • Potential applications in autoimmune conditions through T-cell response modulation

  • Effects on macrophage polarization and inflammatory cytokine production

  • Synergistic effects with existing immunosuppressive therapies

To advance these applications, researchers should focus on:

• Developing tissue-selective FDFT1 inhibitors through targeted delivery systems

• Creating dual-action compounds that inhibit FDFT1 and complementary targets

• Identifying biomarkers to predict responsive patient populations

• Elucidating the precise molecular mechanisms underlying non-cholesterol related effects

These emerging applications position FDFT1 as a versatile therapeutic target beyond its classical role in cholesterol biosynthesis inhibition.

How can structural biology approaches enhance our understanding of FDFT1 function?

Advanced structural biology techniques offer powerful insights into FDFT1 function, mechanism, and inhibitor design. Researchers should consider the following methodological approaches:

The practical applications of these structural insights include:

• Structure-based design of novel FDFT1 inhibitors with improved specificity

• Engineering FDFT1 variants with altered product specificity or enhanced stability

• Elucidation of conformational changes during the catalytic cycle

• Understanding species-specific differences in inhibitor binding

Researchers should prioritize obtaining structures of bovine FDFT1 in multiple functional states to facilitate evolutionary comparisons and enable rational protein engineering approaches.

What are the key considerations for researchers beginning work with recombinant Bovine FDFT1?

Researchers initiating work with recombinant Bovine FDFT1 should consider several critical factors to ensure successful outcomes. This enzyme presents unique challenges due to its membrane association and bifunctional nature, requiring careful methodological planning.

The expression and purification of active FDFT1 demands particular attention to:

• Selection of appropriate expression systems (E. coli BL21(DE3) with pET vectors has proven successful)

• Optimization of culture conditions (reduced temperature post-induction)

• Buffer composition (inclusion of 10% glycerol and 0.4M urea significantly enhances solubility)

• Purification strategy (IMAC followed by additional chromatographic steps)

• Storage conditions (lyophilization or flash-freezing with 6% trehalose)

Activity assays should be carefully designed to account for the two-step reaction mechanism, with appropriate controls and standardized reporting of specific activity. Researchers should anticipate potential challenges in maintaining enzyme stability and develop strategies to distinguish between the two catalytic steps.

Future directions in FDFT1 research hold promise in multiple domains, including structural biology, drug discovery, and metabolic engineering. By adhering to optimized protocols and understanding the intrinsic properties of this enzyme, researchers can make significant contributions to our understanding of sterol biosynthesis and its implications for health and disease.