Recombinant Human Fatty acyl-CoA reductase 2 (FAR2)

What is the primary function of FAR2 in human biochemistry?

FAR2 (Fatty Acyl-CoA Reductase 2) is an enzyme that catalyzes the reduction of saturated and unsaturated fatty acyl-CoA molecules to fatty alcohols. Similar to its paralog FAR1, it plays an essential role in the biosynthesis of ether lipids, plasmalogens, and wax monoesters. The reduction reaction specifically targets C16 or C18 fatty acyl-CoA substrates and requires NADPH as a cofactor. This enzymatic activity represents a critical step in lipid metabolism pathways, particularly in specialized tissues where FAR2 expression is predominant .

How does FAR2 differ from its paralog FAR1 in terms of structure and function?

While FAR2 shares fundamental catalytic mechanisms with FAR1, significant differences exist in their tissue expression patterns, substrate preferences, and regulatory mechanisms. FAR1 is more widely expressed across tissues and primarily localizes to peroxisomal membranes with its N-terminal catalytic domain facing the cytosol and C-terminus exposed to the peroxisomal matrix. FAR2 demonstrates more tissue-specific expression profiles and may have evolved specialized functions related to particular lipid synthesis pathways. Despite these differences, both enzymes maintain the core function of reducing fatty acyl-CoA to fatty alcohols, a critical step in lipid metabolism .

What are the key genetic and protein identifiers for FAR2?

Researchers studying FAR2 should reference the following standardized identifiers:

These identifiers are essential for consistent database searches and cross-referencing in research publications .

What expression systems are most effective for producing recombinant FAR2?

The in vitro E. coli expression system has been successfully employed for recombinant FAR2 production. When designing expression protocols, researchers should consider codon optimization for bacterial expression, as human proteins often contain codons rarely used in E. coli. Additionally, the inclusion of affinity tags (typically His-tag) facilitates efficient purification while maintaining enzymatic activity. For structural studies requiring higher yields, baculovirus expression systems may offer advantages. The choice between prokaryotic and eukaryotic expression systems should be guided by the specific experimental requirements, considering factors such as post-translational modifications and protein folding complexities .

What purification strategies yield the highest activity retention for recombinant FAR2?

Optimal purification strategies balance yield with activity retention. A multi-step approach typically begins with immobilized metal affinity chromatography (IMAC) for His-tagged recombinant FAR2, followed by ion-exchange chromatography to remove impurities. Critical factors affecting enzyme activity include buffer composition (typically 50 mM Tris-HCl, pH 7.5, 150 mM NaCl), reducing agent concentration (1-5 mM DTT or 2-10 mM β-mercaptoethanol), and glycerol content (10-20%). Purification should be conducted at 4°C with protease inhibitors to prevent degradation. The final preparation should be assessed for purity via SDS-PAGE and activity using standardized fatty acyl-CoA reduction assays measuring NADPH consumption spectrophotometrically.

How can researchers effectively measure FAR2 enzymatic activity in vitro?

Enzymatic activity measurement of FAR2 requires careful experimental design. The standard assay monitors the decrease in NADPH absorbance at 340 nm as FAR2 consumes this cofactor during fatty acyl-CoA reduction. A typical reaction mixture contains:

Alternative methods include direct measurement of fatty alcohol production using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), which provide more comprehensive insights into substrate specificity and product formation patterns. For kinetic analyses, researchers should establish Michaelis-Menten parameters by varying substrate concentrations while maintaining excess NADPH .

How do regulatory mechanisms modulate FAR2 activity in different cellular contexts?

FAR2 activity is regulated through multiple mechanisms including transcriptional control, post-translational modifications, and metabolite feedback. At the transcriptional level, lipid-responsive transcription factors likely control expression in a tissue-specific manner. Post-translationally, phosphorylation at specific serine/threonine residues modulates enzymatic activity, with regulatory kinases varying by cell type. Similar to FAR1, FAR2 activity may also be regulated by plasmalogen levels through product inhibition mechanisms. Advanced research should employ phosphoproteomic analyses to identify specific modification sites and metabolomic approaches to understand the relationship between cellular lipid profiles and FAR2 activity regulation .

What methodological approaches best elucidate FAR2's role in specialized lipid biosynthesis pathways?

Comprehensive investigation of FAR2's role requires integrating multiple methodologies:

• CRISPR-Cas9 gene editing to create FAR2 knockout or knock-in cell lines

• Lipidomic profiling using LC-MS/MS to identify specific lipid species affected

• Stable isotope labeling to track metabolic flux through FAR2-dependent pathways

• Proximity labeling techniques (BioID or APEX) to identify protein interaction partners

• Super-resolution microscopy to determine precise subcellular localization

These approaches, when combined, provide a systems-level understanding of FAR2's contribution to specialized lipid biosynthesis. Researchers should be particularly attentive to compensatory mechanisms that may activate when FAR2 function is disrupted, potentially masking phenotypes in single-gene perturbation experiments .

What are the critical considerations when designing FAR2 inhibition studies?

When designing inhibition studies targeting FAR2, researchers must address several methodological challenges:

• Selectivity: Distinguish between FAR1 and FAR2 inhibition, given their structural similarities

• Mechanism: Target either substrate binding, NADPH binding, or allosteric sites

• Cellular permeability: Design compounds with appropriate physicochemical properties

• Off-target effects: Comprehensively profile against related reductases

• Validation methods: Employ enzymatic assays, cellular lipid profiling, and target engagement studies

Successful inhibitor development requires iterative optimization guided by structure-activity relationship studies. Virtual screening approaches using homology models based on FAR1 structures provide starting points for inhibitor design. Researchers should validate hits through multiple orthogonal assays to confirm target specificity and mechanism of action .

How can differential substrate specificity between FAR1 and FAR2 be experimentally determined?

Determining differential substrate specificity requires systematic biochemical analysis using purified recombinant enzymes. Researchers should:

• Express and purify both FAR1 and FAR2 under identical conditions

• Prepare a panel of fatty acyl-CoA substrates varying in:

  • Chain length (C12-C24)

  • Saturation state (saturated, mono-, and polyunsaturated)

  • Branching patterns (straight-chain vs. branched)

• Measure enzyme kinetics (Km, Vmax, kcat) for each substrate

• Calculate specificity constants (kcat/Km) to quantify preference

The following table illustrates typical results from such comparative analyses:

These comparative analyses reveal distinctive substrate preferences that inform physiological roles in specialized lipid biosynthesis pathways .

What methodological approaches best identify tissue-specific expression patterns of FAR2 versus FAR1?

Comprehensive analysis of tissue-specific expression requires multiple complementary approaches:

• Transcriptomic analysis:

  • RNA-Seq across multiple tissues and developmental stages

  • Single-cell RNA-Seq to identify cell-type specificity

  • Quantitative RT-PCR for targeted validation

• Protein-level analysis:

  • Western blotting with isoform-specific antibodies

  • Immunohistochemistry on tissue sections

  • Proteomics with tissue enrichment analysis

• Functional genomics:

  • Reporter gene assays with promoter fragments

  • Chromatin immunoprecipitation to identify transcription factor binding

  • ATAC-Seq to assess chromatin accessibility at gene loci

Integration of these datasets provides a comprehensive map of when and where FAR1 and FAR2 are expressed, informing hypotheses about their specialized functions in different tissues .

What methodologies are most effective for identifying FAR2 mutations in patient samples?

Comprehensive mutation screening requires a multi-tiered approach:

• Next-generation sequencing approaches:

  • Targeted gene panels including FAR2 and related genes

  • Whole exome sequencing with focused analysis of lipid metabolism genes

  • Whole genome sequencing for intronic and regulatory variants

• Variant classification pipeline:

  • In silico prediction tools (SIFT, PolyPhen, CADD)

  • Population frequency databases (gnomAD, 1000 Genomes)

  • Conservation analysis across species

  • Structural modeling of amino acid substitutions

• Functional validation:

  • Site-directed mutagenesis of recombinant FAR2

  • Enzymatic activity assays of mutant proteins

  • Cell-based complementation assays

  • Patient-derived cells for lipidomic profiling

This comprehensive approach ensures rigorous identification and characterization of clinically relevant FAR2 variants .

How can researchers effectively model FAR2 deficiency to understand its pathophysiological consequences?

Developing robust disease models for FAR2 deficiency requires multiple complementary approaches:

• Cellular models:

  • CRISPR-Cas9 knockout in relevant cell types

  • Patient-derived fibroblasts or induced pluripotent stem cells

  • Conditional knockdown systems (shRNA, CRISPRi)

• Animal models:

  • Conventional and conditional knockout mice

  • Humanized mice expressing patient-specific mutations

  • Zebrafish models for high-throughput phenotyping

• Analysis pipeline:

  • Lipidomic profiling focusing on plasmalogens and wax esters

  • Transcriptomic analysis to identify compensatory mechanisms

  • Functional assays relevant to tissues affected in patients

  • Electron microscopy to examine peroxisome morphology