Recombinant Neosartorya fumigata 3-ketoacyl-CoA reductase (AFUA_2G11540)

What is Neosartorya fumigata 3-ketoacyl-CoA reductase and what is its role in fungal metabolism?

Neosartorya fumigata (also known as Aspergillus fumigatus) 3-ketoacyl-CoA reductase (AFUA_2G11540) is a critical enzyme involved in fatty acid elongation pathways. Similar to its homologs in other species, this enzyme likely catalyzes the second step in very-long-chain fatty acid biosynthesis, specifically the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor . The reaction can be represented as:

very-long-chain 3-oxoacyl-CoA + NADPH + H⁺ → very-long-chain (3R)-3-hydroxyacyl-CoA + NADP⁺

In fungi like N. fumigata, this enzyme contributes to the synthesis of lipids essential for cell membrane integrity, virulence factor production, and survival during infection processes . It's particularly important for the organism's adaptability to different host environments during pathogenesis.

How does the structure of N. fumigata 3-ketoacyl-CoA reductase compare to homologous enzymes in other species?

While specific structural data on N. fumigata 3-ketoacyl-CoA reductase is limited in the provided sources, we can draw inferences from homologous enzymes. Based on similar enzymes characterized in other species, N. fumigata 3-ketoacyl-CoA reductase likely belongs to the short-chain dehydrogenase/reductase (SDR) superfamily .

The enzyme likely contains:

• A conserved Rossmann fold for nucleotide binding

• A catalytic triad/tetrad in the active site

• A substrate-binding domain that accommodates very-long-chain fatty acyl substrates

When analyzing structural homology, researchers should consider:

Researchers investigating structural aspects should employ comparative modeling techniques using solved structures of related enzymes, with validation through experimental methods such as circular dichroism or X-ray crystallography.

What are the optimal expression systems for producing recombinant N. fumigata 3-ketoacyl-CoA reductase?

When selecting an expression system for recombinant N. fumigata 3-ketoacyl-CoA reductase, researchers should consider several factors that influence protein yield, folding, and activity. Based on approaches used for similar enzymes, the following systems offer distinct advantages:

For membrane-associated enzymes like 3-ketoacyl-CoA reductase, researchers should consider adding a solubility tag (e.g., MBP, SUMO) or expressing a truncated version excluding transmembrane domains to enhance solubility while maintaining catalytic activity . Expression conditions should be optimized through small-scale pilot experiments before scaling up production.

Researchers should monitor expression through time-course SDS-PAGE analysis and Western blotting, with activity assays to confirm proper folding. This methodological approach ensures the production of functionally active recombinant protein suitable for downstream applications.

What purification strategies yield highest purity and activity for recombinant N. fumigata 3-ketoacyl-CoA reductase?

Purification of recombinant N. fumigata 3-ketoacyl-CoA reductase requires a strategic multi-step approach to maintain enzyme activity while achieving high purity. Based on established protocols for similar enzymes, the following purification workflow is recommended:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag is effective for initial purification. Use a gradient of 20-250 mM imidazole in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and 0.1% appropriate detergent (for membrane-associated forms) .

• Intermediate Purification: Ion exchange chromatography (IEX) on a Q-Sepharose column at pH 7.5-8.0, with elution using a 0-500 mM NaCl gradient.

• Polishing Step: Size exclusion chromatography (Superdex 200) to remove aggregates and achieve final purity.

This process typically yields >95% pure protein as assessed by SDS-PAGE and silver staining. Throughout purification, researchers should:

• Maintain temperature at 4°C to prevent degradation

• Include 1 mM DTT or 5 mM β-mercaptoethanol to protect thiol groups

• Add 10% glycerol to all buffers to enhance stability

• Test enzyme activity after each purification step to monitor functional integrity

Activity assays should measure the NADPH-dependent reduction of 3-ketoacyl-CoA substrates spectrophotometrically by monitoring the decrease in absorbance at 340 nm. Researchers should expect a specific activity of approximately 5-15 μmol/min/mg protein for properly folded enzyme, though this may vary based on substrate chain length.

How should researchers measure the kinetic parameters of N. fumigata 3-ketoacyl-CoA reductase?

Determining accurate kinetic parameters for N. fumigata 3-ketoacyl-CoA reductase requires careful experimental design and consideration of multiple factors. The standard assay employs spectrophotometric measurement of NADPH oxidation at 340 nm . Researchers should implement the following methodological approach:

• Reaction Buffer Optimization:

  • 100 mM potassium phosphate buffer (pH 6.5-7.5)

  • 150 mM NaCl

  • 1 mM DTT or TCEP (reducing agent)

  • Temperature: 25-30°C (optimization required)

• Substrate Preparation:

  • Test various chain-length 3-ketoacyl-CoA substrates (C16-C34)

  • Prepare stock solutions in water or buffer containing 0.1% BSA to prevent substrate adhesion to tubes

  • Use concentration ranges of 1-100 μM for Km determination

• Experimental Design:

  • Standard assay volume: 200 μL in 96-well plate format or 1 mL in cuvette

  • Fixed NADPH concentration (200 μM) when determining substrate Km

  • Fixed substrate concentration (5x Km) when determining NADPH Km

  • Measure initial rates (first 10% of substrate conversion)

  • Include enzyme-free and substrate-free controls

• Data Analysis:

  • Plot initial velocity vs. substrate concentration

  • Fit to Michaelis-Menten equation using non-linear regression

  • Calculate apparent Km, Vmax, kcat, and kcat/Km

For typical very-long-chain 3-ketoacyl-CoA reductases, researchers can expect Km values in the low micromolar range (5-50 μM) for fatty acyl-CoA substrates, with potential variation based on chain length. The enzyme likely follows Michaelis-Menten kinetics, though substrate inhibition may occur at higher concentrations (>100 μM) due to detergent-like effects of acyl-CoA molecules.

What factors most significantly affect the activity and stability of recombinant N. fumigata 3-ketoacyl-CoA reductase?

The activity and stability of recombinant N. fumigata 3-ketoacyl-CoA reductase are influenced by multiple experimental conditions that researchers must carefully control. Based on studies of related enzymes, the following factors have significant impacts:

Researchers have observed that very-long-chain 3-ketoacyl-CoA reductases often exhibit higher stability in the presence of glycerol (10-20%) or other osmolytes. For long-term storage, the enzyme should be maintained at high concentration (>1 mg/mL) in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT at -80°C, with aliquoting to avoid repeated freeze-thaw cycles.

Thermal shift assays (differential scanning fluorimetry) provide a rapid method to screen stabilizing conditions, while long-term activity assays (measuring activity retention over time) should be performed to validate storage conditions.

How does N. fumigata 3-ketoacyl-CoA reductase contribute to fungal virulence and pathogenicity?

N. fumigata 3-ketoacyl-CoA reductase likely plays multiple roles in fungal virulence through its contribution to lipid metabolism. While direct evidence for this specific enzyme is limited in the provided sources, we can extrapolate based on known virulence mechanisms of N. fumigata and the role of fatty acid metabolism in fungal pathogens:

• Cell Wall and Membrane Integrity: Very-long-chain fatty acids synthesized through pathways involving 3-ketoacyl-CoA reductase are essential components of fungal membranes and contribute to cell wall structure. These components affect the organism's resistance to host defense mechanisms, antifungal agents, and environmental stresses .

• Metabolic Adaptation During Infection: N. fumigata can modify its metabolism to adapt to the host environment during infection. The fatty acid elongation pathway likely contributes to this adaptability by allowing the production of specialized lipids needed for survival in different host tissues .

• Secondary Metabolite Production: N. fumigata produces several virulence-associated secondary metabolites, including ergot alkaloids like fumigaclavines that have been shown to contribute significantly to virulence in insect models . The biosynthesis of these compounds may indirectly depend on fatty acid metabolism pathways involving 3-ketoacyl-CoA reductase.

Researchers investigating the relationship between 3-ketoacyl-CoA reductase and virulence should consider:

• Creating knockout or knockdown mutants to assess effects on fatty acid composition and virulence

• Performing lipidomic analysis to identify specific lipid species affected by enzyme dysfunction

• Utilizing infection models (such as Galleria mellonella larvae, similar to studies with ergot alkaloid pathways) to assess virulence differences

• Examining enzyme expression levels during different stages of infection

The interconnection between lipid metabolism and virulence represents an important research direction, as it may reveal potential targets for antifungal development.

What structural features differentiate N. fumigata 3-ketoacyl-CoA reductase from human homologs, and how can these be exploited for antifungal drug development?

Understanding the structural differences between N. fumigata 3-ketoacyl-CoA reductase and human homologs provides valuable insights for selective antifungal drug development. While detailed structural information on the N. fumigata enzyme is not provided in the search results, we can propose the following methodological approach to identify and exploit such differences:

• Comparative Sequence Analysis:

  • Perform multiple sequence alignment between N. fumigata 3-ketoacyl-CoA reductase and human homologs (including human very-long-chain 3-ketoacyl-CoA reductase)

  • Identify regions of low sequence conservation, particularly near the active site and substrate-binding pocket

  • Quantify conservation scores at catalytic residues and substrate-interaction sites

• Homology Modeling and Structural Analysis:

  • Generate homology models based on crystal structures of related enzymes

  • Compare binding pocket architecture and electrostatic surface potential

  • Perform molecular dynamics simulations to identify differences in flexibility and conformational states

• Exploitable Structural Features:
  Based on known differences between fungal and human enzymes in similar pathways, researchers should focus on:

  Structural Feature	Potential Differences	Drug Development Strategy
  Substrate binding pocket	Likely broader in fungal enzyme to accommodate diverse chain lengths	Design inhibitors that exploit unique pocket geometry
  Cofactor binding site	May have different residues interacting with adenosine portion of NADPH	Develop compounds that mimic NADPH but interact with fungal-specific residues
  Allosteric sites	Potential regulatory sites present only in fungal enzyme	Identify and target fungal-specific allosteric sites
  Access channels	Different topology affecting substrate access	Design molecules that block fungal-specific channels

• Inhibitor Design Approaches:

  • Structure-based virtual screening against fungal-specific binding pockets

  • Fragment-based drug discovery focused on differential binding sites

  • Rational design of substrate analogs with substituents directed toward non-conserved regions

  • Development of covalent inhibitors targeting fungal-specific cysteine residues

Researchers have found success with similar approaches targeting other enzymes, such as the development of AKR1C3 inhibitors like ASP9521 . This compound demonstrates how selective targeting of similar enzymes can be achieved through structural understanding. For N. fumigata 3-ketoacyl-CoA reductase, in vitro inhibition assays should evaluate both antifungal activity and mammalian cell toxicity to ensure selective targeting.

What are the most common challenges in expressing active recombinant N. fumigata 3-ketoacyl-CoA reductase, and how can researchers overcome them?

Researchers frequently encounter several challenges when expressing recombinant N. fumigata 3-ketoacyl-CoA reductase. Based on experience with similar enzymes and recombinant protein production, the following methodological solutions are recommended:

For particularly problematic expressions, researchers should consider:

• Cell-Free Expression Systems: These bypass issues related to cell toxicity and provide better control over expression conditions.

• Fungal Expression Hosts: Homologous expression in Aspergillus or Pichia systems may provide native folding machinery specific to fungal enzymes.

• Refolding Protocols: If inclusion bodies are unavoidable, develop a systematic refolding protocol using a matrix approach varying pH, ionic strength, redox conditions, and additives.

• Protein Engineering: Consider expressing individual domains separately or creating chimeric constructs with well-expressed homologs to improve yield while maintaining catalytic function.

Successful expression often requires iterative optimization and combination of multiple strategies tailored to the specific challenges of N. fumigata 3-ketoacyl-CoA reductase.

How can researchers effectively investigate the interaction of N. fumigata 3-ketoacyl-CoA reductase with other components of the fatty acid elongation complex?

Investigating protein-protein interactions (PPIs) within the fatty acid elongation complex requires a multi-technique approach to capture both stable and transient interactions. Based on successful studies of similar multi-component enzyme complexes , researchers should implement the following methodological strategy:

• Co-Immunoprecipitation (Co-IP) Studies:

  • Generate specific antibodies against N. fumigata 3-ketoacyl-CoA reductase or use epitope-tagged versions

  • Perform reciprocal Co-IP experiments to confirm interactions

  • Use mild detergents (0.5-1% NP-40 or 0.1-0.3% digitonin) to preserve membrane protein complexes

  • Analyze by Western blotting with antibodies against suspected interaction partners

  Similar approaches have successfully identified protein-protein interactions in other systems, such as the interaction between TSHR and CD40 proteins demonstrated through Co-IP studies .

• Proximity-Based Labeling:

  • Generate BioID or TurboID fusion constructs with N. fumigata 3-ketoacyl-CoA reductase

  • Express in fungal cells and induce biotinylation

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate hits by directed co-immunoprecipitation

• Crosslinking Mass Spectrometry (XL-MS):

  • Apply chemical crosslinkers (DSS, BS3, or EDC/NHS) to stabilize protein complexes

  • Digest crosslinked complexes and analyze by LC-MS/MS

  • Identify crosslinked peptides using specialized search algorithms

  • Map interaction interfaces to create structural models

• Microscopy-Based Approaches:

  • Perform immunofluorescence co-localization studies

  • Use Förster Resonance Energy Transfer (FRET) to detect direct interactions

  • Implement Bimolecular Fluorescence Complementation (BiFC) to visualize interactions in living cells

• Functional Validation:

  • Generate knockdown/knockout mutants of suspected interaction partners

  • Assess effects on 3-ketoacyl-CoA reductase activity and localization

  • Perform enzyme assays with reconstituted components to verify functional interactions

When interpreting results, researchers should consider that:

• The fungal fatty acid elongation complex likely contains at least four enzymes: 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase, and enoyl-CoA reductase

• Interactions may be substrate-dependent or condition-specific

• The complex may exist in different stoichiometries depending on cellular conditions

This comprehensive approach ensures robust identification and characterization of protein-protein interactions within the elongation complex.

What are the most promising research opportunities for understanding the role of N. fumigata 3-ketoacyl-CoA reductase in antifungal resistance?

The investigation of N. fumigata 3-ketoacyl-CoA reductase in the context of antifungal resistance presents several high-priority research directions. Based on current understanding of fungal resistance mechanisms and lipid metabolism, researchers should consider the following approaches:

• Comparative Expression Analysis:

  • Measure expression levels of 3-ketoacyl-CoA reductase in antifungal-resistant vs. sensitive strains

  • Perform RNA-seq analysis before and after exposure to different antifungal classes

  • Correlate expression changes with changes in lipid composition and antifungal susceptibility

• Genetic Modification Studies:

  • Generate overexpression and knockdown/knockout mutants

  • Assess impact on susceptibility to multiple antifungal classes (azoles, echinocandins, polyenes)

  • Measure changes in membrane fluidity, permeability, and ergosterol content

• Lipidomic Profiling:

  • Compare lipid profiles between wild-type and mutant strains

  • Identify specific lipid species altered in resistant isolates

  • Correlate changes in very-long-chain fatty acids with resistance phenotypes

• Structure-Function Analysis:

  • Identify natural variants of the enzyme in resistant clinical isolates

  • Perform site-directed mutagenesis to assess the impact of specific residues

  • Develop structure-based models to explain how mutations affect enzyme function

• Inhibitor Development and Testing:

  • Design and screen specific inhibitors targeting N. fumigata 3-ketoacyl-CoA reductase

  • Test combinations with existing antifungals for synergistic effects

  • Evaluate inhibitors against resistant clinical isolates

The connection to antifungal resistance is particularly relevant given that alterations in membrane composition can affect drug uptake, efflux pump activity, and target accessibility. Neosartorya species have demonstrated variable susceptibilities to antifungal drugs, with some showing resistance to multiple agents . Understanding how 3-ketoacyl-CoA reductase contributes to these resistance phenotypes could lead to novel therapeutic strategies or resistance prediction markers.

How might systems biology approaches enhance our understanding of N. fumigata 3-ketoacyl-CoA reductase in fungal metabolism and pathogenesis?

Systems biology offers powerful frameworks to comprehensively understand the role of N. fumigata 3-ketoacyl-CoA reductase within the context of broader metabolic networks and pathogenesis mechanisms. Researchers should consider implementing the following methodological approaches:

• Multi-Omics Integration:

  • Combine transcriptomics, proteomics, and lipidomics data

  • Apply network analysis to identify functional modules connected to 3-ketoacyl-CoA reductase

  • Develop predictive models of metabolic flux through the fatty acid elongation pathway

  • Experimental design should include multiple growth conditions, host interaction models, and antifungal exposures

• Genome-Scale Metabolic Modeling:

  • Incorporate 3-ketoacyl-CoA reductase into existing N. fumigata metabolic models

  • Perform flux balance analysis to predict metabolic consequences of enzyme perturbation

  • Identify synthetic lethal interactions that could be exploited for combination therapies

  • Validate model predictions through targeted metabolomics experiments

• Host-Pathogen Interaction Networks:

  • Map changes in fungal metabolism during different stages of infection

  • Identify how 3-ketoacyl-CoA reductase activity changes in response to host factors

  • Develop dual RNA-seq approaches to simultaneously track host and pathogen responses

  • Connect enzyme activity to specific virulence phenotypes observed in infection models

• Temporal and Spatial Dynamics:

  • Implement time-course experiments to track metabolic changes during growth and infection

  • Use fluorescent reporters to monitor enzyme localization and activity in living cells

  • Apply microfluidics and single-cell approaches to capture heterogeneity in enzyme expression

  • Correlate enzyme dynamics with changes in cell morphology and virulence factor production

• Comparative Systems Analysis:

  • Compare metabolic networks across multiple Aspergillus and Neosartorya species

  • Identify conserved and divergent roles of 3-ketoacyl-CoA reductase

  • Correlate network differences with variations in pathogenicity

  • Apply evolutionary systems biology to understand how the enzyme's role has evolved

This systems approach would help contextualize the finding that N. fumigata produces various virulence factors, including ergot alkaloids and other specialized metabolites that contribute to pathogenesis . Understanding how 3-ketoacyl-CoA reductase fits within these broader metabolic networks would provide insights into potential intervention points and the fundamental biology of this important pathogen.