Recombinant Chicken Fatty acyl-CoA reductase 1 (FAR1)

What is chicken Fatty acyl-CoA reductase 1 (FAR1) and what is its function?

Chicken Fatty acyl-CoA reductase 1 (FAR1) is an enzyme that catalyzes the reduction of fatty acyl-CoA thioesters to fatty alcohols. This enzyme plays a critical role in the biosynthesis pathway of wax esters, which are components of the uropygial gland secretions in birds. The primary function of FAR1 is to produce fatty alcohols by reducing the thioester bond in fatty acyl-CoA using NADPH as an electron donor .

In chicken (Gallus gallus domesticus), FAR1 is one of two FAR isoforms (FAR1 and FAR2) that have been identified and characterized. The enzymatic activity of FAR1 leads to the production of not only fatty alcohols but also fatty aldehydes as intermediates, although the proportion of aldehydes varies depending on the substrate chain length .

How does chicken FAR1 differ from FAR2 and similar enzymes in other avian species?

Chicken FAR1 differs from FAR2 and other avian FAR enzymes in several key aspects:

Substrate preference:

• Chicken FAR1 (GgFAR1) shows higher activity with C16:0-CoA compared to C14:0-CoA and C18:0-CoA

• Chicken FAR2 (GgFAR2) demonstrates highest activity with C18:0-CoA

pH optimum:

• FAR1 enzymes typically have a pH optimum around 6.5

• FAR2 enzymes display highest activity at approximately pH 5.5

Aldehyde production:

• FAR1 enzymes produce distinctly higher aldehyde levels than FAR2 enzymes

• Aldehyde production varies with the chain length of the acyl-CoA and is highest in FAR1 assays with C14:0-CoA

When comparing across avian species, barn owl (Tyto alba) FAR1 (TaFAR1) shows the highest activity in yeast expression systems, producing up to 18 μmol fatty alcohol per gram fresh weight. The chain length specificity increases from goose (Anser domesticus) AdFAR1, via chicken GgFAR1 to barn owl TaFAR1, while chicken GgFAR2 has a more pronounced substrate specificity than barn owl TaFAR2 .

What are the optimal conditions for recombinant chicken FAR1 enzymatic activity?

Based on experimental studies with membrane fractions containing recombinant chicken FAR1, the optimal conditions for enzymatic activity include:

Cofactor requirements:

• NADPH is required as an electron donor

• Optimal NADPH concentration is approximately 5 mM

Temperature and reaction time:

• Enzymatic activities remain constant for up to 15 minutes at 37°C

pH conditions:

• FAR1 has an optimal pH of approximately 6.5

Substrate requirements:

• Activated acyl groups (acyl-CoAs) are required as substrates

• Addition of ATP and CoA to free fatty acids restores FAR activity, confirming the requirement for activated substrates

Cellular localization:

• Activity is detectable only in the total membrane fraction, consistent with the predicted transmembrane domain in the C-terminal region of avian FAR proteins

These parameters are essential considerations when designing experimental protocols for recombinant chicken FAR1 enzymatic assays.

What expression systems are optimal for producing functional recombinant chicken FAR1?

Yeast expression systems have proven effective for the functional production of recombinant chicken FAR1. Based on the experimental evidence available, the following methodology has been successfully employed:

Yeast expression system advantages:

• Provides a eukaryotic cellular environment suitable for proper folding of membrane-associated proteins like FAR1

• Allows for functional studies directly in intact cells through feeding experiments with exogenous fatty acids

• Enables subcellular fractionation for enzyme characterization studies

Expression validation:

• Functional expression of chicken FAR1 in yeast results in the production of fatty alcohols, which can be detected and quantified to confirm enzymatic activity

• The catalytic properties of the avian sequences can be verified through both in vivo fatty alcohol production in intact cells and in vitro enzyme assays with membrane fractions

While the search results don't specifically address other expression systems, it's important to note that membrane-associated proteins like FAR1 often present challenges in bacterial expression systems like E. coli due to potential issues with proper folding and membrane insertion. Though not mentioned in the available search results for FAR1 specifically, other avian recombinant proteins have been successfully expressed in E. coli, as demonstrated by the expression of chicken spike protein fragments in the third search result .

How can researchers assess the enzymatic activity of recombinant chicken FAR1?

Researchers can assess the enzymatic activity of recombinant chicken FAR1 using multiple complementary approaches:

In vivo activity in transgenic yeast cells:

• Culture transgenic yeast expressing chicken FAR1

• Extract lipids from yeast cells

• Analyze fatty alcohol content through appropriate analytical methods (e.g., GC-MS)

• Compare fatty alcohol profiles with control yeast (transformed with empty vector)

Feeding experiments:

• Supplement transgenic yeast cultures with specific fatty acids of interest

• Harvest cells and extract lipids

• Analyze resulting fatty alcohol profiles to determine substrate utilization patterns in vivo

• This approach has been successfully used to test the ability of chicken FAR1 to utilize odd-numbered, even-numbered, and monounsaturated fatty acids

In vitro enzyme assays:

• Prepare subcellular fractions (total membrane fraction) from FAR1-expressing yeast

• Conduct enzyme assays with specific acyl-CoA substrates and NADPH

• Measure the production of fatty alcohols and/or aldehydes

• Typical assay conditions include appropriate pH buffer (pH 6.5 for FAR1), 5 mM NADPH, and defined acyl-CoA concentrations

• Reactions are typically conducted at 37°C for up to 15 minutes to ensure linear reaction rates

Analytical considerations:

• Multiple analytical methods can be employed to detect and quantify fatty alcohols and aldehydes

• Direct comparison with control samples is essential to account for background activity

• When studying chain length specificity, using a range of acyl-CoA substrates (C10 to C22) provides comprehensive activity profiles

What purification strategies are effective for isolating recombinant chicken FAR1 while maintaining enzymatic activity?

While the search results don't provide specific purification protocols for chicken FAR1, important considerations can be inferred from the enzymatic characteristics described:

Membrane protein considerations:

• FAR1 activity is associated exclusively with membrane fractions, indicating it is a membrane-bound enzyme

• This is consistent with the predicted transmembrane domain in the C-terminal region of avian FAR proteins

• Purification strategies must account for this membrane association

Subcellular fractionation:

• Initial separation of cellular components through differential centrifugation to isolate membrane fractions

• The total membrane fraction containing FAR1 activity can be prepared from transgenic yeast cells expressing the recombinant protein

Potential affinity purification approaches:
Although not explicitly mentioned for chicken FAR1, general recombinant protein techniques that might be applicable include:

• Addition of affinity tags (His-tag, etc.) for affinity chromatography

• Use of detergents that maintain enzymatic activity while solubilizing membrane proteins

• Careful optimization of buffer conditions to maintain the pH optimum of FAR1 (approximately 6.5)

These considerations highlight the importance of maintaining the membrane environment or finding suitable detergent conditions for FAR1 purification while preserving enzymatic activity.

What is the substrate specificity profile of chicken FAR1 compared to other avian FAR enzymes?

Chicken FAR1 (GgFAR1) displays distinct substrate preferences that differentiate it from other avian FAR enzymes. Detailed comparative analysis reveals:

Straight-chain saturated fatty acyl-CoA preferences:

• GgFAR1 shows highest activity with C16:0-CoA

• GgFAR1 has moderate activity with C14:0-CoA and C18:0-CoA

• GgFAR1 shows considerably lower activity with short-chain substrates (C10:0-CoA and C12:0-CoA)

Comparative analysis across species:

• Barn owl FAR1 (TaFAR1) shows similar substrate preferences to chicken FAR1, with highest activity toward C16:0-CoA

• Goose FAR1 (AdFAR1) demonstrates a more relaxed chain length specificity, with almost equal activity across C14:0-CoA, C16:0-CoA, and C18:0-CoA

• In contrast, FAR2 enzymes from both chicken and barn owl show highest activity with C18:0-CoA

Branched-chain fatty acyl-CoA utilization:

Odd-numbered fatty acyl-CoA utilization:

• When supplied with odd-numbered fatty acids (C13:0 to C19:0), yeast expressing GgFAR1 produces significant amounts of C15:0-OH (up to 65% of total alcohols)

• C17:0-OH is also produced but in lower quantities than C15:0-OH and C16:0-OH

This substrate specificity profile indicates that chicken FAR1 has evolved to preferentially process medium-chain (C16) fatty acyl-CoAs, distinguishing it functionally from both FAR2 enzymes and FAR1 enzymes from other avian species.

How does chicken FAR1 process branched-chain fatty acyl-CoAs compared to straight-chain substrates?

Chicken FAR1 demonstrates significantly different processing capabilities for branched-chain fatty acyl-CoAs compared to straight-chain substrates:

Activity with 2-methyl-branched substrates:

Lack of activity with multi-methyl-branched substrates:

• No reductase activity was detected with phytanoyl-CoA (3,7,11,15-tetramethyl-C16-CoA)

• This suggests that multiple methyl branches significantly impair substrate recognition or processing by chicken FAR1

Aldehyde formation differences:

• With straight-chain substrates, GgFAR1 produces detectable amounts of fatty aldehydes as reaction intermediates

• In contrast, aldehyde synthesis was not observed with branched-chain substrates

• This may indicate altered reaction kinetics or substrate binding with branched-chain substrates

Biological implications:

• The low activity with branched-chain substrates suggests that chicken FAR1 would produce branched-chain fatty alcohols only if provided with an acyl-CoA pool largely consisting of branched-chain acyl groups

• This is consistent with the observation that chicken uropygial glands produce wax esters without branched-chain alcohols, while barn owl glands produce wax esters containing branched-chain alcohols despite having FAR enzymes with very similar properties

These findings indicate that the presence of methyl branches significantly impacts substrate processing by chicken FAR1, with implications for the composition of naturally produced wax esters.

What kinetic parameters characterize chicken FAR1 enzymatic activity?

While the search results don't provide comprehensive kinetic parameters (Km, Vmax, kcat) for chicken FAR1, several important kinetic characteristics can be extracted:

NADPH dependency:

• Chicken FAR1 requires NADPH as an electron donor

• The optimal NADPH concentration is approximately 5 mM

• This suggests a Km for NADPH in the low millimolar range

Reaction time course:

• Enzymatic activities remain constant for up to 15 minutes at 37°C

• This indicates that under appropriate assay conditions, the reaction maintains linearity for at least 15 minutes, which is important for accurate determination of initial velocities

Substrate utilization patterns:

• Chicken FAR1 shows different activities with various acyl-CoA substrates

• Highest activity is observed with C16:0-CoA

• Relative activities with different chain lengths provide insights into substrate preferences, though specific Km values are not reported

Aldehyde formation:

• FAR1 enzymes produce aldehydes as reaction intermediates

• The proportion of aldehydes varies with substrate chain length, being highest with C14:0-CoA

• This suggests that the second reduction step (aldehyde to alcohol) may be rate-limiting under certain conditions

pH dependency:

• Chicken FAR1 has a pH optimum of approximately 6.5

• This pH dependency provides insights into the catalytic mechanism and the protonation states of key catalytic residues

For more precise kinetic characterization, researchers would need to conduct detailed enzyme kinetic studies with purified recombinant chicken FAR1, measuring initial velocities at varying substrate concentrations to determine Km, Vmax, and kcat values.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of chicken FAR1?

Site-directed mutagenesis offers a powerful approach to investigate the catalytic mechanism of chicken FAR1, although specific mutagenesis studies are not described in the search results. Based on the enzymatic characteristics and general principles of FAR enzymes, the following strategies would be valuable:

Target residues for mutagenesis:

• NADPH binding site residues: Mutations in the predicted NADPH binding domain could alter cofactor affinity and help identify critical residues for electron transfer

• Catalytic residues: FAR enzymes typically contain conserved catalytic residues that participate in the reduction reaction

• Substrate binding pocket residues: Mutations in regions that interact with the acyl chain could alter chain length specificity or activity with branched-chain substrates

• Membrane-association domain: Modifications to the C-terminal transmembrane domain could provide insights into the importance of membrane association for activity

Functional analyses of mutants:

• Express mutant proteins in yeast systems as described for wild-type FAR1

• Compare fatty alcohol production profiles in vivo

• Conduct in vitro enzyme assays with membrane fractions containing mutant proteins

• Assess changes in substrate specificity, pH optimum, and aldehyde/alcohol ratios

Investigating the two-step reduction mechanism:

• Since FAR1 produces both aldehydes and alcohols, mutations could potentially separate these activities

• Targeted mutations might create variants that accumulate aldehydes without completing the reduction to alcohols

• This would provide insights into the two-step reduction mechanism (acyl-CoA → aldehyde → alcohol)

Comparison with FAR2 isoform:

• Creating chimeric proteins between FAR1 and FAR2 could help identify domains responsible for their different substrate preferences and pH optima

• Such chimeras could reveal structural determinants of the higher aldehyde production observed with FAR1 compared to FAR2

These approaches would significantly advance understanding of the structure-function relationships in chicken FAR1 and the molecular basis of its catalytic properties.

What experimental approaches can be used to investigate the membrane association of chicken FAR1?

Given that chicken FAR1 activity is detected exclusively in membrane fractions, understanding its membrane association is crucial. Although the search results don't describe specific membrane association studies for FAR1, several experimental approaches would be valuable:

Bioinformatic analysis:

• Computational prediction of transmembrane domains and membrane-association motifs

• The search results mention a predicted transmembrane domain in the C-terminal region of avian FAR proteins

• Hydropathy plots and secondary structure predictions can further characterize these domains

Truncation and deletion studies:

• Generate C-terminal truncations removing the predicted transmembrane domain

• Express these truncated variants in yeast systems

• Assess their subcellular localization and enzymatic activity

• Determine whether membrane association is essential for catalytic function

Subcellular fractionation and localization:

• Perform detailed subcellular fractionation of yeast cells expressing chicken FAR1

• Use marker proteins to identify specific membrane compartments (ER, Golgi, plasma membrane)

• Determine the precise subcellular localization of FAR1 activity

• This would provide insights into the membrane environment where FAR1 naturally functions

Detergent solubilization studies:

• Test various detergents for their ability to solubilize active FAR1 from membranes

• Identify conditions that maintain enzymatic activity in a solubilized state

• This approach could facilitate purification strategies and provide insights into lipid requirements for activity

Reconstitution experiments:

• Incorporate solubilized FAR1 into artificial membrane systems (liposomes, nanodiscs)

• Test whether specific lipid compositions affect enzymatic activity

• This would reveal potential lipid requirements or preferences for optimal FAR1 function

These experimental approaches would provide a comprehensive understanding of the membrane association of chicken FAR1 and its importance for enzymatic function.

How might structural modeling and comparative analysis inform our understanding of chicken FAR1?

While the search results don't discuss structural aspects of chicken FAR1, structural modeling and comparative analysis would provide valuable insights:

Homology modeling approaches:

• Identify suitable template structures from related enzymes in protein structure databases

• Generate homology models of chicken FAR1 using computational tools

• Validate models through energy minimization and structural evaluation

• Use models to predict substrate binding sites and catalytic residues

Structural basis for substrate specificity:

• Compare structural models of chicken FAR1 and FAR2 to identify differences in substrate binding pockets

• This could explain the observed preference of FAR1 for C16:0-CoA versus the preference of FAR2 for C18:0-CoA

• Analyze the binding pocket dimensions and chemical properties that might accommodate different chain lengths

Cross-species comparison:

• Generate structural models for FAR1 enzymes from different bird species (chicken, barn owl, goose)

• Compare these models to identify structural features that might explain functional differences

• For example, identify structural elements that might account for the more relaxed chain length specificity of goose FAR1 compared to chicken FAR1

Structure-guided mutagenesis:

• Use structural models to identify residues potentially involved in:

  • NADPH binding

  • Acyl-CoA substrate binding

  • Catalysis of the two reduction steps

  • Membrane association

• These predictions could guide targeted mutagenesis experiments to test structure-function hypotheses

Molecular dynamics simulations:

• Perform molecular dynamics simulations of FAR1 models in membrane environments

• Investigate protein-membrane interactions and their effects on protein dynamics

• Simulate substrate binding and potential conformational changes during catalysis

These computational approaches, combined with experimental validation, would significantly advance understanding of chicken FAR1 structure-function relationships.

What is the biological role of chicken FAR1 in uropygial gland function?

The biological role of chicken FAR1 in uropygial gland function can be understood in the context of wax ester biosynthesis:

Contribution to wax ester synthesis:

• Chicken FAR1 catalyzes the production of fatty alcohols, which are essential components of wax esters in uropygial gland secretions

• These wax esters contribute to the waterproofing and maintenance of feathers

• By producing primarily straight-chain fatty alcohols with chain lengths of C16 and C18, chicken FAR1 directly influences the composition of these wax esters

Relationship with uropygial gland wax ester composition:

• Chicken uropygial glands produce wax esters without branched-chain alcohols

• This is consistent with the low activity of chicken FAR1 with branched-chain substrates

• The substrate specificity of FAR1 thus appears to match the natural composition of wax esters in the uropygial gland secretions

Coordinated function with other enzymes:

• FAR1 functions within a broader metabolic pathway including fatty acid synthesis and wax ester formation

• The search results indicate that uropygial glands control their synthesis of multi-branched fatty acids by regulating the substrate pool available to fatty acid synthetase

• Similarly, the composition of the acyl-CoA pool available to FAR1 likely influences the alcohol composition of wax esters

Complementary roles of FAR1 and FAR2:

• The presence of two FAR isoforms (FAR1 and FAR2) with different substrate preferences suggests complementary roles

• Together, they may ensure production of a diverse range of fatty alcohols for incorporation into wax esters

• Their different pH optima might allow regulation of their relative activities under different physiological conditions

Understanding the biological role of chicken FAR1 in uropygial gland function provides insights into the molecular basis of bird feather maintenance and waterproofing mechanisms.

How do the enzymatic properties of chicken FAR1 compare to FAR enzymes in mammals and other organisms?

The search results provide insights into the comparison between avian and mammalian FAR enzymes:

Substrate specificity differences:

• Avian FAR1 enzymes (including chicken FAR1) prefer medium-chain substrates (C16:0-CoA)

• In contrast, mammalian FAR enzymes generally show different acyl-CoA specificities

• Avian FAR2 enzymes resemble mammalian isozymes in their preference for C18 acyl groups

Evolutionary implications:

• The differential substrate preferences between avian and mammalian FAR enzymes suggest evolutionary adaptation to different physiological requirements

• These adaptations likely reflect the different compositions of wax esters required for feathers versus mammalian skin and fur

Functional conservation:

• Despite differences in substrate specificity, the basic enzymatic function (reduction of acyl-CoAs to fatty alcohols) is conserved across species

• This suggests that FAR enzymes evolved from a common ancestral enzyme while adapting to specific physiological needs in different lineages

Membrane association:

• The membrane association of chicken FAR1 appears to be a conserved feature across FAR enzymes

• This suggests that membrane localization is important for the proper function of these enzymes across diverse organisms

A more comprehensive comparison would require additional data about FAR enzymes from other organisms, but the available information highlights both conserved features and species-specific adaptations in these enzymes.

What insights can be gained from comparing FAR1 enzymes across different avian species?

Comparative analysis of FAR1 enzymes from different avian species provides valuable insights into evolutionary adaptation and functional specialization:

Species-specific substrate preferences:

Correlation with wax ester composition:

• These differences in substrate specificity correlate with species-specific compositions of uropygial gland secretions

• For example, barn owl uropygial glands produce wax esters with branched-chain alcohols, while chicken and goose glands produce wax esters without such alcohols

• This suggests evolutionary adaptation of FAR enzymes to meet species-specific requirements for feather maintenance

Enzymatic activity variations:

• TaFAR1 from barn owl showed the highest activity in yeast expression systems

• The chain length specificity increases in the order: AdFAR1 (goose) < GgFAR1 (chicken) < TaFAR1 (barn owl)

• These variations may reflect different selective pressures related to habitat, behavior, or other ecological factors

Evolutionary adaptation mechanisms:

These comparative insights illuminate the evolutionary adaptation of FAR1 enzymes across avian species and provide context for understanding their functional specialization.