Recombinant Neosartorya fumigata 3-ketoacyl-CoA reductase (AFUB_027290)

What is Neosartorya fumigata 3-ketoacyl-CoA reductase and what is its function?

Neosartorya fumigata 3-ketoacyl-CoA reductase (AFUB_027290) is an enzyme involved in fatty acid metabolism in the pathogenic fungus Neosartorya fumigata (also known as Aspergillus fumigatus). The enzyme belongs to the short-chain dehydrogenase/reductase (SDR) family and catalyzes the reduction of 3-ketoacyl-CoA intermediates to 3-hydroxyacyl-CoA in the fatty acid elongation cycle. This reaction represents the second step in fatty acid biosynthesis, which is essential for membrane lipid formation and fungal growth. The enzyme utilizes NADPH as a cofactor for the reduction reaction and is characterized by its EC number 1.1.1.- .

What are the alternative names and classifications for this enzyme?

The enzyme is known by several alternative names:

• 3-ketoacyl-CoA reductase

• 3-ketoreductase

• KAR

• Microsomal beta-keto-reductase

Its gene name is AFUB_027290, and its UniProt accession number is B0XSI3. The enzyme is classified under EC 1.1.1.- (oxidoreductases acting on the CH-OH group of donors with NAD+ or NADP+ as acceptor) .

How can I express recombinant Neosartorya fumigata 3-ketoacyl-CoA reductase in a laboratory setting?

For recombinant expression of Neosartorya fumigata 3-ketoacyl-CoA reductase, the following methodological approach is recommended:

• Gene Amplification: PCR-amplify the AFUB_027290 gene (coding for amino acids 1-345) using genomic DNA from Neosartorya fumigata strain CEA10/CBS 144.89/FGSC A1163 as template.

• Expression Vector Construction: Clone the amplified gene into an appropriate expression vector (pET system vectors work well for heterologous protein expression). Include a purification tag (His6, GST, or FLAG) to facilitate downstream purification.

• Host Selection: Transform the construct into a suitable E. coli expression strain such as BL21(DE3), Rosetta, or Origami for expression. For more complex folding requirements, consider Pichia pastoris or insect cell expression systems.

• Expression Conditions: Optimize expression by testing:

  • Induction temperature (16-37°C)

  • IPTG concentration (0.1-1.0 mM)

  • Expression duration (4-24 hours)

  • Media composition (LB, TB, or auto-induction media)

• Solubility Assessment: Analyze protein solubility by SDS-PAGE comparison of whole-cell lysate, soluble, and insoluble fractions.

For fungal membrane proteins like 3-ketoacyl-CoA reductase, expression may require optimization to ensure proper folding and activity retention.

What purification methods are effective for isolating Neosartorya fumigata 3-ketoacyl-CoA reductase?

A multi-step purification strategy is recommended:

• Initial Capture:

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

  • For GST-tagged constructs: Glutathione affinity chromatography

• Intermediate Purification:

  • Ion exchange chromatography (IEX): Based on the theoretical pI of the protein

  • Hydrophobic interaction chromatography (HIC): Particularly useful for separating properly folded from misfolded species

• Polishing Step:

  • Size exclusion chromatography (SEC): To obtain highly pure monomeric protein and determine oligomeric state

• Buffer Optimization:

  • Standard buffer: 50 mM Tris-HCl pH 7.5-8.0, 150-300 mM NaCl, 5-10% glycerol

  • Consider including stabilizing additives: 0.5-1 mM DTT or 2-mercaptoethanol, 0.1 mM EDTA

• Storage Conditions:

  • For short-term: 4°C in optimized buffer

  • For long-term: -20°C or -80°C in 50% glycerol-containing buffer to prevent freezing damage

How can I assess the enzymatic activity of Neosartorya fumigata 3-ketoacyl-CoA reductase?

The activity of 3-ketoacyl-CoA reductase can be measured using several complementary approaches:

• Spectrophotometric Assay:

  • Principle: Monitor the oxidation of NADPH at 340 nm (decrease in absorbance)

  • Reaction mixture components:

    • 100 mM Tris-HCl buffer (pH 7.5)

    • 0.15 mM NADPH

    • 0.1-0.5 mM 3-ketoacyl-CoA substrate (acetoacetyl-CoA is commonly used)

    • Purified enzyme (0.1-1 μg)

  • Calculate activity using the extinction coefficient of NADPH (ε = 6220 M⁻¹cm⁻¹)

• HPLC-based Product Analysis:

  • Separate substrate and product using reverse-phase HPLC

  • Use C18 column with acetonitrile/water gradient

  • Monitor CoA derivatives at 254-260 nm

• Coupled Enzyme Assay:

  • Couple the production of NADP+ to a secondary enzyme reaction that produces a more easily detectable signal

• Controls and Validation:

  • Negative control: Reaction without enzyme

  • Positive control: Commercial 3-ketoacyl-CoA reductase from related species

  • Inhibition control: Include known SDR inhibitors (e.g., NADP+ analogs)

How does Neosartorya fumigata 3-ketoacyl-CoA reductase contribute to fungal pathogenicity?

The role of 3-ketoacyl-CoA reductase in fungal pathogenicity is multifaceted and encompasses several interconnected mechanisms:

• Membrane Integrity and Adaptation:

  • The enzyme is crucial for fatty acid biosynthesis, which directly affects membrane composition

  • Proper membrane fluidity is essential for surviving host environmental stresses (temperature, pH, oxidative stress)

  • Altered membrane composition can affect virulence factor secretion

• Biofilm Formation:

  • Fatty acid metabolism impacts extracellular matrix production

  • Disruption of fatty acid biosynthesis genes in related fungi has been shown to impair biofilm formation and reduce virulence

• Host-Pathogen Interaction:

  • Fatty acid-derived molecules serve as signaling compounds during infection

  • These compounds can modulate host immune responses

• Potential as Drug Target:

  • The distinctiveness of fungal fatty acid metabolism compared to human pathways makes it an attractive antifungal target

  • Inhibition of 3-ketoacyl-CoA reductase could potentially disrupt multiple virulence mechanisms simultaneously

What molecular mechanisms determine substrate specificity in Neosartorya fumigata 3-ketoacyl-CoA reductase?

The substrate specificity of Neosartorya fumigata 3-ketoacyl-CoA reductase is governed by several structural determinants:

• Substrate Binding Pocket Architecture:

  • The enzyme possesses a hydrophobic tunnel that accommodates the acyl chain

  • Key residues forming this pocket include conserved leucine, isoleucine, and phenylalanine residues

  • The length and shape of this pocket determine chain length preference

• Catalytic Residues:

  • The catalytic tetrad (Asn-Ser-Tyr-Lys) positions the substrate optimally for hydride transfer

  • The Tyr residue acts as a proton donor in the reaction mechanism

  • The Ser and Asn residues form a hydrogen-bonding network stabilizing reaction intermediates

• Cofactor Binding Region:

  • The Rossmann fold region binds NADPH with high specificity

  • Residues that interact with the 2'-phosphate group of NADPH determine the preference for NADPH over NADH

• Conformational Dynamics:

  • The enzyme undergoes conformational changes upon binding of NADPH

  • These changes prepare the active site for substrate binding

  • Further conformational adjustments occur during the catalytic cycle

A detailed understanding of these mechanisms requires crystallographic studies or homology modeling based on related enzymes, combined with site-directed mutagenesis to confirm the role of specific residues.

How can genetic modification techniques be utilized to study Neosartorya fumigata 3-ketoacyl-CoA reductase function?

Several genetic approaches can be employed to investigate the function of 3-ketoacyl-CoA reductase in Neosartorya fumigata:

• Gene Knockout/Deletion:

  • Create a knockout construct by replacing part of the AFUB_027290 coding sequence with a selection marker (e.g., hygromycin resistance gene)

  • Use homologous recombination for targeted integration

  • Verify successful deletion by PCR and Southern blotting

  • Analyze the phenotypic consequences, including growth, morphology, and virulence

• Gene Complementation:

  • Reintroduce the wild-type AFUB_027290 gene into the knockout strain

  • Confirm phenotypic restoration to validate the specific role of the enzyme

• Site-Directed Mutagenesis:

  • Introduce specific mutations in catalytic or substrate-binding residues

  • Assess the effects on enzyme activity and substrate specificity

  • Generate structure-function relationships

• Conditional Expression Systems:

  • Place the gene under an inducible promoter

  • Study the immediate consequences of enzyme depletion

  • Useful for essential genes where knockout might be lethal

• Reporter Gene Fusion:

  • Create GFP or other fluorescent protein fusions

  • Determine subcellular localization and expression patterns

  • Monitor enzyme dynamics in living cells

These approaches, particularly gene knockout and complementation studies as demonstrated in the analysis of other fungal enzymes like EasM, provide powerful tools for understanding protein function in vivo .

What analytical methods can be used to characterize substrate specificity of Neosartorya fumigata 3-ketoacyl-CoA reductase?

To characterize the substrate specificity of Neosartorya fumigata 3-ketoacyl-CoA reductase, researchers can employ the following analytical approaches:

• Steady-State Kinetics:

  • Determine Km and kcat values for various 3-ketoacyl-CoA substrates of different chain lengths

  • Calculate catalytic efficiency (kcat/Km) to quantify preference

  • Data can be organized in a table format:

• Isothermal Titration Calorimetry (ITC):

  • Measure binding affinity (Kd) for different substrates

  • Determine thermodynamic parameters (ΔH, ΔG, ΔS)

  • Provide insights into the binding mechanism

• Mass Spectrometry-Based Approaches:

  • Use LC-MS/MS to identify and quantify reaction products

  • Perform competitive assays with substrate mixtures to determine preferences

  • Monitor time-course of substrate conversion for complex substrate mixtures

• Structural Analysis:

  • X-ray crystallography with bound substrates or substrate analogs

  • Molecular docking simulations to predict binding modes

  • Molecular dynamics simulations to study enzyme-substrate interactions

How can I resolve contradictory kinetic data for Neosartorya fumigata 3-ketoacyl-CoA reductase?

When facing contradictory kinetic data for 3-ketoacyl-CoA reductase, consider these methodological approaches:

• Standardize Experimental Conditions:

  • Ensure consistent buffer composition, pH, and temperature across experiments

  • Verify enzyme purity and stability under assay conditions

  • Standardize substrate preparation methods to eliminate concentration inconsistencies

• Identify Potential Sources of Discrepancy:

  • Enzyme preparation differences (tag position, purification method)

  • Substrate quality and solubility issues

  • Experimental approach variations (direct vs. coupled assays)

  • Presence of inhibitors or activators in reagents

• Systematic Validation Studies:

  • Perform enzyme concentration dependency tests to confirm linearity

  • Conduct time-course studies to ensure initial velocity conditions

  • Evaluate potential product inhibition effects

  • Consider allosteric regulation by testing various substrate concentrations

• Alternative Data Analysis Approaches:

  • Apply different kinetic models (Michaelis-Menten, Hill, etc.)

  • Use global fitting for complex kinetic schemes

  • Perform replicate experiments with statistical analysis

  • Consider ensemble averaging of multiple experimental approaches

• Collaborative Cross-Validation:

  • Have different laboratory members independently perform assays

  • Exchange enzyme preparations or substrates with collaborating labs

  • Consider round-robin testing if discrepancies persist

How does Neosartorya fumigata 3-ketoacyl-CoA reductase compare to similar enzymes in other fungi?

Comparing Neosartorya fumigata 3-ketoacyl-CoA reductase with homologous enzymes from other fungi reveals important evolutionary and functional relationships:

What typing methods can be used to analyze Neosartorya fumigata strains expressing different variants of 3-ketoacyl-CoA reductase?

For characterizing Neosartorya fumigata strains based on 3-ketoacyl-CoA reductase variants, several molecular typing approaches are available:

• Short Tandem Repeat (STR) Analysis:

  • STR assays can discriminate between different Neosartorya fumigata strains with high resolution

  • The standard STR Aspergillus fumigatus (STR Af) typing method uses nine microsatellite markers amplified by multiplex PCR

  • This approach has high discriminatory power (D = 0.994+) and can distinguish closely related isolates

• TRESP (Tandem Repeats within Exons of Surface Protein genes) Typing:

  • Recently developed method based on hypervariable tandem repeats within exons of surface protein coding genes

  • Uses three to four markers for strain discrimination

  • Has comparable discriminatory power to STR but requires less specialized equipment

  • Particularly useful for grouping strains with similar resistance mechanisms

• Combined Approaches for Enhanced Resolution:

  • Integrating STR and TRESP provides improved discriminatory power

  • Removing highly variable markers (like STR Af M3) can sometimes provide more biologically meaningful clustering

  • Combined approach is particularly valuable for epidemiological investigations

• Application to 3-ketoacyl-CoA Reductase Variants:

  • Sequencing the AFUB_027290 gene in different isolates

  • Analyzing expression levels by RT-qPCR

  • Correlating genetic variations with phenotypic characteristics

  • Examining potential associations between reductase variants and antifungal resistance

These methods have been successfully applied to analyze Aspergillus fumigata populations, particularly in the context of antifungal resistance mechanisms, and could be adapted to study 3-ketoacyl-CoA reductase variants .

What are the most promising avenues for targeting Neosartorya fumigata 3-ketoacyl-CoA reductase in antifungal development?

Future research on Neosartorya fumigata 3-ketoacyl-CoA reductase offers several promising directions for antifungal development:

• Structure-Based Drug Design:

  • Determine the crystal structure of Neosartorya fumigata 3-ketoacyl-CoA reductase

  • Identify unique structural features that differentiate it from human homologs

  • Design selective inhibitors targeting fungal-specific binding pockets

  • Develop transition-state analogs as potent inhibitors

• Combination Therapy Approaches:

  • Investigate synergistic effects between 3-ketoacyl-CoA reductase inhibitors and existing antifungals

  • Target multiple steps in the fatty acid biosynthesis pathway simultaneously

  • Exploit metabolic vulnerabilities created by enzyme inhibition

• Alternative Inhibition Strategies:

  • Develop allosteric inhibitors that affect enzyme dynamics rather than active site binding

  • Explore covalent inhibitors that form irreversible bonds with key residues

  • Investigate RNA-based approaches to downregulate enzyme expression

• Biomarker Development:

  • Identify unique metabolic signatures associated with 3-ketoacyl-CoA reductase activity

  • Develop diagnostic tools based on these signatures

  • Enable personalized treatment approaches based on fungal metabolic profiling

• Resistance Mechanism Characterization:

  • Study potential resistance mechanisms that could emerge against 3-ketoacyl-CoA reductase inhibitors

  • Design pre-emptive strategies to overcome predicted resistance mechanisms

  • Implement evolutionary studies to predict resistance development pathways

These research directions have the potential to yield novel antifungal approaches that could address the growing problem of resistance to current antifungal agents.

How might systems biology approaches enhance our understanding of Neosartorya fumigata 3-ketoacyl-CoA reductase in fungal metabolism?

Systems biology offers powerful approaches to contextualize the role of 3-ketoacyl-CoA reductase within the broader metabolic network of Neosartorya fumigata:

• Metabolic Flux Analysis:

  • Use isotope labeling to track carbon flow through fatty acid biosynthesis

  • Quantify how 3-ketoacyl-CoA reductase activity influences global metabolic fluxes

  • Identify potential metabolic chokepoints and compensatory pathways

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map how genetic or environmental perturbations affect 3-ketoacyl-CoA reductase expression and activity

  • Identify regulatory networks controlling enzyme expression and activity

• Genome-Scale Metabolic Modeling:

  • Develop computational models of Neosartorya fumigata metabolism

  • Simulate the effects of 3-ketoacyl-CoA reductase inhibition on fungal growth

  • Predict metabolic vulnerabilities and potential drug targets

• Protein-Protein Interaction Networks:

  • Identify interaction partners of 3-ketoacyl-CoA reductase

  • Map the enzyme's position within multi-protein complexes

  • Understand how physical interactions regulate enzyme function

• Environmental Response Mapping:

  • Characterize how environmental stresses affect 3-ketoacyl-CoA reductase activity

  • Understand enzyme regulation during host infection

  • Identify conditions that might enhance or reduce antifungal efficacy

These systems-level approaches would provide a comprehensive understanding of how 3-ketoacyl-CoA reductase functions within the complex metabolic network of Neosartorya fumigata, potentially revealing unexpected therapeutic opportunities.