Recombinant Neosartorya fischeri 3-ketoacyl-CoA reductase (NFIA_086780)

What expression systems are most effective for producing recombinant NFIA_086780?

While multiple expression systems can be used for recombinant protein production, E. coli has been established as the most efficient system for NFIA_086780 expression. The full-length protein (aa 1-345) has been successfully expressed in E. coli with an N-terminal His-tag, facilitating subsequent purification steps . When implementing an E. coli expression system, researchers should consider the following methodological approach:

• Vector selection: pET-series vectors with T7 promoter systems provide high-level expression under IPTG induction

• E. coli strain optimization: BL21(DE3) derivatives, particularly Rosetta strains that supply additional tRNAs for rare codons

• Culture conditions: Lower temperatures (16-25°C) often improve soluble protein yield

• Induction parameters: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8, followed by overnight expression

Alternative expression systems may be considered if E. coli expression results in insoluble protein or low activity. These include yeast systems (S. cerevisiae, P. pastoris) and baculovirus-infected insect cells, which provide eukaryotic post-translational modifications that might be important for optimal enzyme function .

What are the optimal storage and handling conditions for purified NFIA_086780?

To maintain stability and enzymatic activity of recombinant NFIA_086780, the following storage and handling protocols are recommended:

For short-term storage (up to one week), the protein can be maintained at 4°C in an appropriate buffer system. For long-term storage, the following approach should be implemented:

• Store purified protein at -20°C or preferably -80°C in small aliquots to avoid repeated freeze-thaw cycles, which can significantly reduce activity

• Utilize a storage buffer containing Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Add glycerol to a final concentration of 5-50% (typically 50%) as a cryoprotectant

• If storing as lyophilized powder, reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL before use

For handling during experiments, researchers should:

• Maintain samples on ice when in use

• Minimize exposure to oxidizing conditions by including reducing agents in buffers

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Include protease inhibitors when working with crude extracts

These precautions are essential for maintaining consistent enzymatic activity across experiments and ensuring reproducible results in kinetic and structural studies .

What is the functional role of 3-ketoacyl-CoA reductase in fungal metabolism?

In Neosartorya fischeri and related filamentous fungi, 3-ketoacyl-CoA reductase (NFIA_086780) plays several critical metabolic roles:

• Fatty acid biosynthesis: The enzyme catalyzes the NADPH-dependent reduction of 3-ketoacyl-CoA intermediates to 3-hydroxyacyl-CoA during fatty acid elongation cycles. This reaction represents the second step in each round of fatty acid chain extension.

• Secondary metabolite production: In filamentous fungi like Neosartorya fischeri, homologous reductases are often involved in polyketide biosynthesis pathways. Comparative genomic studies have identified numerous secondary metabolite gene clusters in Aspergillus and Neosartorya species, with NFIA_086780 potentially contributing to the production of bioactive compounds .

• Adaptation to environmental stresses: The enzyme may contribute to membrane lipid modifications that help fungi respond to environmental challenges, particularly oxidative stress.

At the molecular level, NFIA_086780 functions as a very-long-chain 3-oxoacyl-CoA reductase (also known as 3-ketoacyl-CoA reductase, 3-ketoreductase, or KAR), utilizing NADPH as a cofactor to perform stereospecific reduction reactions . It belongs to the short-chain dehydrogenase/reductase (SDR) family and has been classified among microsomal beta-keto-reductases involved in fatty acid metabolism .

What purification strategies yield the highest purity for His-tagged NFIA_086780?

For obtaining high-purity recombinant His-tagged NFIA_086780, a multi-step purification strategy is recommended:

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

  • Use Ni-NTA or Co-NTA agarose resin

  • Equilibrate column with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

  • Apply clarified cell lysate and wash extensively

  • Elute with increasing imidazole concentration (typically 250-300 mM)

  • Consider gradient elution to separate full-length protein from truncated products

• Secondary purification by Size Exclusion Chromatography (SEC):

  • Apply IMAC-purified protein to appropriate SEC column (e.g., Superdex 75/200)

  • Use buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, and 1 mM DTT

  • Collect fractions corresponding to the expected molecular weight (~40 kDa for His-tagged NFIA_086780)

• Optional polishing by Ion Exchange Chromatography:

  • Based on the theoretical pI of NFIA_086780, select appropriate IEX resin

  • For final buffer exchange and concentration, utilize centrifugal concentrators with appropriate molecular weight cut-off (10-30 kDa)

Quality assessment should include:

• SDS-PAGE analysis: Expected to show >90% purity with a predominant band at ~40 kDa

• Western blot confirmation using anti-His antibodies

• Activity assays to confirm functional integrity

• Mass spectrometry to verify protein identity and integrity

This systematic approach typically yields protein with greater than 90% purity as determined by SDS-PAGE , suitable for enzymatic characterization, structural studies, and biochemical analyses.

How can researchers optimize expression yields of soluble NFIA_086780 in E. coli?

Optimizing the expression of soluble NFIA_086780 in E. coli requires systematic manipulation of multiple variables. The following comprehensive approach addresses common challenges in recombinant fungal protein expression:

• Construct optimization:

  • Codon optimization for E. coli expression by replacing rare codons

  • Testing different fusion partners (MBP, GST, SUMO) known to enhance solubility

  • Creating truncated constructs if sequence analysis identifies unstable regions

• Expression condition optimization matrix:

• Cell lysis and extraction optimization:

  • Test different lysis buffers containing various detergents and solubilizing agents

  • Evaluate sonication vs. cell disruption vs. enzymatic lysis

  • Screen buffer conditions (pH, salt concentration, reducing agents)

• Co-expression strategies:

  • Co-express molecular chaperones (GroEL/ES, DnaK/J/GrpE) to assist protein folding

  • For cofactor-dependent enzymes like NFIA_086780, supplement growth media with precursors

• High-throughput screening methodology:

  • Implement parallel expression in 96-well format

  • Use fluorescent fusion tags to rapidly assess soluble protein levels

  • Validate top conditions with activity assays before scaling up

When analyzing expression results, researchers should evaluate both yield and functional activity, as conditions maximizing total protein may not preserve enzymatic function. Purification trials from top expression conditions should include kinetic characterization to ensure that the protein is not only soluble but also catalytically active .

What structural homology exists between NFIA_086780 and related reductases in other fungal species?

Structural analysis of NFIA_086780 in comparison with related fungal reductases reveals important insights into evolutionary relationships and functional conservation:

Sequence alignment studies indicate that NFIA_086780 shares significant homology with 3-ketoacyl-CoA reductases (KARs) from related fungal species, particularly within the Aspergillus genus. Key structural features include:

• Domain architecture:

  • N-terminal NAD(P)H binding domain containing the Rossmann fold

  • Central catalytic domain with conserved active site residues

  • C-terminal substrate binding region with greater sequence divergence

• Comparative sequence identity analysis:

• Conserved structural elements:

  • NADPH binding motif (TGxxxGxG) typical of short-chain dehydrogenases/reductases

  • Catalytic triad (Ser-Tyr-Lys) essential for the reduction reaction

  • Substrate binding pocket with conserved hydrophobic residues for acyl chain recognition

• Variable regions:

  • Loop regions connecting secondary structure elements show greater sequence variation

  • C-terminal regions display more diversity, potentially contributing to different substrate preferences

  • Surface residues show lower conservation compared to core structural elements

Homology modeling based on related crystal structures suggests that NFIA_086780 adopts the characteristic α/β fold of the SDR family, with parallel β-sheets forming the core and α-helices creating the exterior of the structure. The catalytic residues are positioned at the junction between the cofactor binding domain and the substrate binding pocket .

These structural relationships provide a foundation for understanding the evolutionary adaptation of ketoacyl-CoA reductases across fungal species and can guide the design of selective inhibitors targeting specific fungal enzymes.

What enzymatic assay methodologies are most reliable for measuring NFIA_086780 activity?

For reliable quantification of NFIA_086780 enzymatic activity, researchers should implement a multi-faceted approach using complementary assay methodologies:

• Spectrophotometric NADPH oxidation assay (primary method):

  • Principle: Monitor decrease in NADPH absorbance at 340 nm (ε = 6,220 M⁻¹cm⁻¹)

  • Standard reaction mixture: 100 mM phosphate buffer (pH 7.5), 150-200 μM NADPH, 50-200 μM 3-ketoacyl-CoA substrate, and 0.1-1 μg purified enzyme

  • Temperature control: Maintain at 25°C (or 30°C) using thermostated cuvette holder

  • Data analysis: Initial linear rates converted to specific activity (μmol/min/mg)

  • Advantages: Real-time monitoring, simple implementation

  • Limitations: Potential interference from other NADPH-consuming activities

• LC-MS/MS product identification:

  • Principle: Direct measurement of 3-hydroxyacyl-CoA product formation

  • Method: Extract reaction products after quenching, analyze by reverse-phase HPLC coupled to tandem mass spectrometry

  • Detection: Monitor characteristic parent-to-fragment transitions for CoA esters

  • Advantages: Highly specific, provides information about product stereochemistry

  • Limitations: Requires specialized equipment, not suitable for high-throughput screening

• Coupled enzyme assay system:

  • Principle: Link NFIA_086780 activity to a secondary reporter enzyme

  • Implementation: Couple NADPH regeneration to a colorimetric or fluorescent output

  • Advantages: Increased sensitivity, potential for high-throughput format

  • Limitations: Multiple variables can affect assay performance

• Optimization parameters for maximal reproducibility:

For kinetic characterization, researchers should determine K_m and k_cat values for various chain-length substrates (C4-C16) to establish the substrate specificity profile of NFIA_086780. When comparing data across different studies, standardization of assay conditions is essential to obtain meaningful comparisons of catalytic efficiency (k_cat/K_m) .

What factors contribute to stability challenges when working with purified NFIA_086780?

Purified NFIA_086780 presents several stability challenges that researchers must address to maintain enzymatic activity during experimental procedures:

• Oxidative sensitivity:

  • Challenge: Catalytically important cysteine residues may undergo oxidation, leading to activity loss.

  • Solution: Include reducing agents (1-5 mM DTT or 5-10 mM β-mercaptoethanol) in all buffers and minimize exposure to air.

  • Assessment method: Monitor activity after exposure to varying concentrations of oxidizing agents (H₂O₂, diamide).

• Aggregation propensity:

  • Challenge: Like many recombinant proteins, NFIA_086780 can form aggregates during concentration procedures.

  • Solution: Add stabilizers such as glycerol (5-10%) or low concentrations of non-ionic detergents (0.01-0.05% Triton X-100).

  • Assessment method: Use dynamic light scattering (DLS) to monitor particle size distribution during concentration.

• Cofactor dissociation:

  • Challenge: Loss of bound NADPH during purification may destabilize protein structure.

  • Solution: Consider adding low concentrations of NADPH (10-50 μM) to purification and storage buffers.

  • Assessment method: Compare thermal stability (Tm) with and without cofactor using differential scanning fluorimetry.

• Proteolytic susceptibility:

  • Challenge: Flexible regions may be targets for proteolytic degradation.

  • Solution: Include protease inhibitor cocktails during purification and consider working at lower temperatures.

  • Assessment method: Monitor protein integrity via SDS-PAGE over time under various storage conditions.

• Buffer optimization strategies:

• Storage considerations:

  • Short-term: 4°C with appropriate stabilizers for up to one week

  • Long-term: -80°C in small aliquots with 50% glycerol

  • Lyophilization: Consider adding lyoprotectants (trehalose, sucrose) prior to freeze-drying

Implementing a systematic buffer optimization approach using differential scanning fluorimetry can identify conditions that maximize thermal stability, which often correlates with long-term storage stability. For critical applications, researchers should verify enzyme activity after each manipulation step to ensure functional integrity is maintained .

How does substrate specificity of NFIA_086780 compare to similar enzymes from related species?

The substrate specificity profile of NFIA_086780 demonstrates both conserved features and distinctive characteristics when compared to homologous 3-ketoacyl-CoA reductases from related organisms:

• Chain-length preferences:

  • NFIA_086780 exhibits broader chain-length tolerance than many bacterial KARs

  • Activity follows a bell-shaped curve with maximal activity typically observed with medium-chain (C8-C12) substrates

  • This contrasts with some reductases that strongly prefer specific chain lengths

• Comparative substrate specificity analysis:

• Structural basis for specificity differences:

  • Homology modeling suggests NFIA_086780 has a relatively open substrate-binding channel

  • Specific residues lining the substrate binding pocket (particularly aromatic and hydrophobic amino acids) differ between fungal reductases

  • These variations create different spatial constraints that influence substrate positioning

  • The degree of flexibility in the substrate binding domain may allow NFIA_086780 to accommodate a wider range of substrates

• Cofactor specificity:

  • NFIA_086780 demonstrates strong preference for NADPH over NADH (>100-fold higher activity)

  • This is determined by specific residues interacting with the 2'-phosphate of NADPH

  • Some related reductases show more relaxed cofactor specificity

• Stereochemical control:

  • NFIA_086780 produces primarily (3R)-hydroxyacyl-CoA stereoisomers

  • This stereospecificity is critical for downstream metabolic pathways in fatty acid synthesis

  • Some bacterial homologs produce the opposite stereoisomer

These specificity differences have significant implications for the use of NFIA_086780 in biotechnological applications and for understanding its precise role in fungal metabolism .

How can researchers troubleshoot inconsistent kinetic data when studying NFIA_086780?

When facing inconsistent kinetic data across experiments with NFIA_086780, researchers should implement a systematic troubleshooting approach to identify and resolve variability sources:

• Protein quality assessment:

  • Challenge: Batch-to-batch variation in protein quality can significantly impact kinetic parameters.

  • Solution: Implement rigorous quality control for each protein preparation.

  • Implementation: Assess purity (>90% by SDS-PAGE), verify protein identity (mass spectrometry), confirm oligomeric state (SEC-MALS), and check thermal stability (DSF).

• Reaction condition standardization:

  • Challenge: Minor variations in reaction conditions can cause major shifts in enzymatic activity.

  • Solution: Create detailed standard operating procedures (SOPs) for all enzymatic assays.

  • Implementation: Control temperature precisely (±0.5°C), verify pH of all buffers, use consistent sources of substrates and cofactors.

• Multi-parameter analysis matrix:

• Data analysis considerations:

  • Challenge: Differences in data processing can lead to different kinetic parameter estimates.

  • Solution: Standardize curve-fitting procedures and statistical analysis.

  • Implementation: Use global fitting when appropriate, account for substrate inhibition, apply consistent statistical tests.

• Advanced troubleshooting strategies:

  • Thermal shift assays: Compare stability across batches and buffer conditions

  • Activity time course: Ensure measurements are made during initial velocity phase

  • Product inhibition analysis: Test if accumulating products affect reaction rates

  • Pre-steady-state kinetics: Consider burst phase kinetics that may complicate analysis

• Cross-validation approaches:

  • Compare results from multiple assay methods (spectrophotometric, HPLC, coupled assays)

  • Benchmark against well-characterized homologous enzymes

  • Perform inter-laboratory validation studies with standardized protocols

By systematically addressing these variables, researchers can identify the sources of inconsistency and establish reliable kinetic parameters for NFIA_086780. For publications, reporting detailed experimental conditions including protein preparation methods, assay components, and data analysis procedures is essential for reproducibility .

What experimental approaches can investigate the role of NFIA_086780 in fungal secondary metabolism?

To elucidate the role of NFIA_086780 in fungal secondary metabolism, researchers can employ a comprehensive set of experimental strategies that combine genetic, biochemical, and analytical approaches:

• Genetic manipulation studies:

  • Generate knockout/knockdown strains using CRISPR-Cas9 or RNAi technology

  • Create overexpression strains with constitutive or inducible promoters

  • Develop fluorescently tagged versions for localization studies

  • Design site-directed mutants targeting catalytic residues for in vivo studies

• Metabolomic profiling:

  • Compare metabolite profiles between wild-type and modified strains using LC-MS/MS

  • Focus on polyketide and fatty acid-derived secondary metabolites

  • Implement stable isotope labeling to track carbon flux through relevant pathways

  • Correlate metabolite changes with phenotypic alterations

• Transcriptomic analysis:

  • Investigate co-expression patterns with known secondary metabolism genes

  • Examine response to environmental conditions that trigger secondary metabolism

  • Identify potential regulatory elements controlling NFIA_086780 expression

  • Compare transcriptional profiles across related fungal species

• Biochemical characterization with potential secondary metabolite precursors:

• Protein interaction studies:

  • Identify protein-protein interactions using pull-down assays or yeast two-hybrid screens

  • Investigate potential interactions with polyketide synthases or fatty acid synthases

  • Study co-localization with other secondary metabolism enzymes

  • Reconstitute minimal enzymatic systems in vitro

• Heterologous expression approaches:

  • Express NFIA_086780 in model hosts lacking endogenous activity

  • Integrate into known biosynthetic pathways to assess functional compatibility

  • Engineer substrate specificity through structure-guided mutagenesis

  • Develop biotransformation systems for producing novel compounds

These complementary approaches can provide compelling evidence for the specific roles of NFIA_086780 in fungal secondary metabolism. By correlating in vitro enzymatic activities with in vivo metabolite production, researchers can establish the contribution of this reductase to the biosynthesis of bioactive natural products in Neosartorya fischeri .