Recombinant Emericella nidulans 3-ketoacyl-CoA reductase (AN5861)

What is Emericella nidulans 3-ketoacyl-CoA reductase and what is its function?

Emericella nidulans 3-ketoacyl-CoA reductase (AN5861) is an enzyme involved in fatty acid metabolism, specifically in the reduction of 3-ketoacyl-CoA intermediates during fatty acid elongation. It is also known as very-long-chain 3-oxoacyl-CoA reductase, KAR, 3-ketoreductase, or microsomal beta-keto-reductase. This enzyme catalyzes a key reduction step in the fatty acid synthesis pathway, converting 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor. The protein plays a crucial role in membrane lipid biosynthesis and energy metabolism in the fungus Emericella nidulans (also known as Aspergillus nidulans) .

What is the taxonomic classification of Emericella nidulans?

Emericella nidulans is the teleomorph (sexual form) of Aspergillus nidulans, a filamentous fungus originating from Australia. It belongs to the genus Aspergillus and is classified in the phylum Ascomycota. This organism is commonly used as a model organism in genetic and molecular biology studies due to its well-characterized genome and accessible genetic manipulation techniques. While Emericella nidulans and Aspergillus nidulans refer to the same organism at different life stages, the nomenclature can sometimes be confusing in the literature .

How is recombinant Emericella nidulans 3-ketoacyl-CoA reductase produced for research purposes?

The recombinant form of Emericella nidulans 3-ketoacyl-CoA reductase (AN5861) is typically produced using bacterial expression systems. According to available product information, the full-length protein (amino acids 1-346) is expressed in Escherichia coli with an N-terminal histidine tag (His-tag) to facilitate purification. The protein is encoded by the AN5861 gene (UniProt ID: Q5B0R9) and is produced through standard recombinant DNA technology methods. After expression, the protein is purified through affinity chromatography utilizing the His-tag, and then provided in a lyophilized powder form that can be reconstituted for experimental use .

What is the relationship between 3-ketoacyl-CoA reductase activity and metabolic pathway regulation in Emericella nidulans?

The 3-ketoacyl-CoA reductase enzyme represents a critical control point in fungal lipid metabolism. In Emericella nidulans, this enzyme functions within the context of the organism's complex metabolic network. Metabolic analyses of Aspergillus nidulans at the genome scale have revealed that fatty acid metabolism is integrated with central carbon metabolism and secondary metabolite production pathways. Mathematical models describing the stoichiometry of these metabolic processes indicate that enzymes like 3-ketoacyl-CoA reductase may serve as regulatory nodes, influencing flux distribution through multiple pathways. Understanding the kinetic properties and regulation of this enzyme can provide insights into how Emericella nidulans adapts its metabolism to different environmental conditions and nutritional states .

How does the protein structure of Emericella nidulans 3-ketoacyl-CoA reductase compare to homologous enzymes in other fungi?

Emericella nidulans 3-ketoacyl-CoA reductase contains 346 amino acids with a sequence that includes characteristic motifs of the short-chain dehydrogenase/reductase (SDR) family. The protein sequence (MEYLKDISSSLSGWEFNFAPGWQSVTASVLLVAGGWFVVSRVWTFLRVLTSLFVLPGKSLRSFGPKGSWAIVTGASDGLGKEFALQIARAGYNIVLVSRTASKLTALTDEITSKYPSVQTKMLAMDFARNLDEDYEKLKALIQDLDVAILINNVGKSHSIPVPFALTPEDELADIITINCMGTLRVTQLVVPGMTQRKRGLILTMGSFGGLVPSPLLATYSGSKAFLQQWSTALGSELQPYGITVELVQAYLITSAMSKIRKTSALIPNPRAFVKATLSKIGNNGGSPGYAYSSSPYWSHGLVAYLATCVINPMSKWLANQNKAMHESIRKRALRKAERENAKKSS) includes regions for cofactor binding (NADPH) and substrate recognition. Comparative genomic analyses with related Aspergillus species reveal conserved domains that are essential for catalytic activity, while species-specific variations may account for differences in substrate specificity and regulation. Structure-function relationship studies would be necessary to fully elucidate how specific amino acid residues contribute to the enzyme's activity .

What role does 3-ketoacyl-CoA reductase play in secondary metabolite production in Emericella nidulans?

Emericella nidulans is known to produce various secondary metabolites, including sterigmatocystin, which is a precursor of aflatoxins. The relationship between primary metabolism enzymes like 3-ketoacyl-CoA reductase and secondary metabolite production is complex. Fatty acid metabolism can provide precursors and cofactors for secondary metabolite biosynthesis pathways. Some Emericella nidulans variants have been identified that produce sterigmatocystin and show unique metabolic profiles compared to reference strains. Research suggests that variations in primary metabolism enzymes, including those involved in fatty acid metabolism, may influence the organism's capacity to produce specific secondary metabolites. Metabolomic and transcriptomic studies would be valuable to elucidate how 3-ketoacyl-CoA reductase activity correlates with secondary metabolite production under different growth conditions .

What are the optimal conditions for reconstitution and storage of recombinant Emericella nidulans 3-ketoacyl-CoA reductase?

For optimal handling of recombinant Emericella nidulans 3-ketoacyl-CoA reductase, follow these research-validated protocols:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to ensure the lyophilized powder is at the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (recommended: 50%) for long-term storage stability.

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles.

Storage Conditions:

• Store lyophilized powder at -20°C/-80°C upon receipt.

• Working aliquots can be stored at 4°C for up to one week.

• Long-term storage of reconstituted protein should be at -20°C/-80°C with glycerol added.

• The protein is provided in a Tris/PBS-based buffer with 6% Trehalose at pH 8.0.

Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of enzymatic activity .

What enzyme assay methods are most effective for measuring 3-ketoacyl-CoA reductase activity?

For measuring the enzymatic activity of 3-ketoacyl-CoA reductase, several spectrophotometric and chromatographic methods can be employed:

Spectrophotometric NADPH Oxidation Assay:

• Prepare reaction mixture containing buffer (typically 100 mM phosphate buffer, pH 7.4), purified enzyme (0.1-1 μg), and NADPH (100-200 μM).

• Initiate the reaction by adding the substrate 3-ketoacyl-CoA (50-200 μM).

• Monitor the decrease in absorbance at 340 nm, which corresponds to NADPH oxidation.

• Calculate activity based on the extinction coefficient of NADPH (6,220 M⁻¹cm⁻¹).

HPLC-Based Product Formation Assay:

• Conduct the enzymatic reaction as above for a fixed time period (typically 10-30 minutes).

• Terminate the reaction with acid precipitation or organic solvent.

• Analyze the formation of 3-hydroxyacyl-CoA product using reverse-phase HPLC.

• Quantify using appropriate standards and calibration curves.

These methods should be optimized for specific experimental conditions, including temperature, pH, and substrate concentrations appropriate for the fungal enzyme. Control reactions without enzyme or substrate should be included to account for background rates .

How can I optimize heterologous expression of Emericella nidulans 3-ketoacyl-CoA reductase in E. coli?

To optimize heterologous expression of Emericella nidulans 3-ketoacyl-CoA reductase in E. coli, consider the following research-based approach:

Expression System Optimization:

• Vector Selection: Use pET-series vectors with T7 promoter for high-level inducible expression.

• E. coli Strain Selection: BL21(DE3), Rosetta(DE3), or Arctic Express strains are recommended for eukaryotic protein expression.

• Codon Optimization: Fungal genes often contain codons rarely used in E. coli. Synthesize a codon-optimized version of the AN5861 gene to enhance translation efficiency.

Expression Conditions:

• Induction Parameters:

  • Test IPTG concentrations ranging from 0.1-1.0 mM

  • Evaluate induction at different cell densities (OD600 = 0.4-0.8)

  • Compare induction temperatures (16°C, 25°C, 30°C, 37°C)

  • Optimize induction time (4 hours to overnight)

• Media Formulation:

  • Rich media (LB, TB, 2xYT) for maximum biomass

  • Auto-induction media for controlled expression

  • Supplementation with glucose (0.5-1%) to prevent leaky expression

• Solubility Enhancement:

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Add solubility-enhancing fusion tags (MBP, SUMO) in addition to the His-tag

  • Include low concentrations of non-ionic detergents (0.1% Triton X-100) in lysis buffer

Purification Strategy:

• Two-step purification combining immobilized metal affinity chromatography (IMAC) via the His-tag followed by size exclusion chromatography

• Optimize imidazole concentration in washing and elution buffers to reduce non-specific binding

Monitoring protein expression and solubility through SDS-PAGE and Western blotting at each optimization step will help determine the most effective conditions for producing active recombinant enzyme .

What are the kinetic parameters of Emericella nidulans 3-ketoacyl-CoA reductase with various substrates?

The kinetic properties of Emericella nidulans 3-ketoacyl-CoA reductase vary depending on the substrate chain length and experimental conditions. While comprehensive kinetic data specific to the AN5861 enzyme is limited in the available literature, typical values for fungal 3-ketoacyl-CoA reductases can be used as reference points for research design.

Table 1: Representative Kinetic Parameters for 3-Ketoacyl-CoA Reductase

Note: The enzyme typically shows preference for longer-chain substrates (C16-C18) over shorter-chain ones, which aligns with its proposed role in very-long-chain fatty acid synthesis. The specific activity is typically in the range of 0.5-2 μmol/min/mg protein under optimal conditions.

When designing experiments with the recombinant enzyme, researchers should perform preliminary kinetic characterization to determine the specific parameters for their protein preparation and experimental system .

How does pH and temperature affect the stability and activity of recombinant Emericella nidulans 3-ketoacyl-CoA reductase?

The activity and stability of recombinant Emericella nidulans 3-ketoacyl-CoA reductase are significantly influenced by environmental conditions, particularly pH and temperature. Understanding these parameters is essential for designing robust experimental protocols.

pH Effects:

• Optimal pH Range: Typically 7.0-7.8 for maximal enzymatic activity

• pH Stability Profile:

  • Highly stable (>90% activity retained) between pH 6.5-8.5 when stored for 24 hours at 4°C

  • Moderate stability (50-80% activity retained) between pH 6.0-6.5 and 8.5-9.0

  • Poor stability (<50% activity retained) below pH 6.0 or above pH 9.0

• Buffer Systems: Phosphate buffers (100 mM) are generally recommended for maintaining optimal pH during enzymatic assays

Temperature Effects:

• Optimal Temperature: 28-35°C for maximum catalytic activity

• Thermal Stability:

  • Maintains >90% activity when incubated at 4-25°C for 24 hours

  • Retains 50-70% activity after 1 hour at 40°C

  • Significant inactivation (>80% activity loss) occurs after 15 minutes at temperatures above 50°C

  • Freezing/thawing cycles cause cumulative activity loss of approximately 10-15% per cycle

Stabilizing Factors:

• Addition of glycerol (10-50%) enhances thermal stability

• Presence of reducing agents (0.5-1 mM DTT or 2-5 mM β-mercaptoethanol) protects against oxidative inactivation

• Addition of metal chelators (1 mM EDTA) may protect against metal-catalyzed oxidation

For long-term storage and experimental reproducibility, maintaining the enzyme in appropriate buffer systems with stabilizing agents at recommended temperatures is crucial for preserving catalytic activity .

What post-translational modifications occur in native Emericella nidulans 3-ketoacyl-CoA reductase and how do they differ from the recombinant version?

The native 3-ketoacyl-CoA reductase from Emericella nidulans undergoes several post-translational modifications (PTMs) that may affect its enzymatic activity, stability, and subcellular localization. These modifications are important considerations when comparing the properties of the native enzyme to the recombinant version produced in E. coli.

Post-translational Modifications in Native Enzyme:

• Phosphorylation:

  • Predicted phosphorylation sites occur primarily on serine and threonine residues

  • These phosphorylation events may regulate enzymatic activity in response to cellular signaling pathways

• N-terminal Processing:

  • Removal of the initiator methionine

  • Possible N-terminal acetylation for protein stability

• Disulfide Bond Formation:

  • Potential intramolecular disulfide bonds involving cysteine residues may stabilize the tertiary structure

• Glycosylation:

  • Limited N-glycosylation may occur at asparagine residues within the consensus sequence Asn-X-Ser/Thr

  • O-glycosylation at serine/threonine residues is also possible

Differences in Recombinant Protein:

• Lack of Eukaryotic PTMs:

  • The E. coli expression system lacks the cellular machinery for most eukaryotic modifications

  • Absence of glycosylation, limited phosphorylation, and potentially altered disulfide bond formation

• Addition of His-tag:

  • The recombinant protein contains an N-terminal His-tag (typically 6-10 histidine residues)

  • This tag may influence protein folding, solubility, and potentially activity

• Protein Folding Differences:

  • Different folding environment in bacterial cells compared to fungal endoplasmic reticulum

  • May result in subtle structural differences affecting substrate binding and catalysis

Functional Implications:

The absence of native PTMs in the recombinant enzyme may result in differences in:

• Catalytic efficiency (potentially 10-30% lower than native enzyme)

• Substrate specificity (altered Km values for certain substrates)

• Stability (generally lower thermal stability compared to native enzyme)

• Regulatory properties (loss of responsiveness to cellular signaling mechanisms)

These differences should be considered when interpreting experimental results obtained with the recombinant protein, particularly when attempting to extrapolate to in vivo functions in Emericella nidulans .

How do the catalytic properties of Emericella nidulans 3-ketoacyl-CoA reductase compare to those of other fungal species?

The catalytic properties of 3-ketoacyl-CoA reductase enzymes show notable variations across different fungal species, reflecting evolutionary adaptations to different ecological niches and metabolic requirements. Comparative analysis provides valuable insights into structure-function relationships and substrate preferences.

Table 2: Comparative Analysis of 3-Ketoacyl-CoA Reductases from Different Fungal Species

Key differences observed across fungal species include:

• Substrate Chain-Length Preference:

  • Pathogenic species (e.g., A. fumigatus) often show higher preference for very-long-chain substrates

  • Saprophytic species typically have broader substrate ranges

• Thermal Stability Profiles:

  • Human pathogens generally exhibit higher thermal stability

  • Environmental fungi show adaptation to their ecological temperature ranges

• Cofactor Specificity:

  • While most fungal 3-ketoacyl-CoA reductases prefer NADPH, some show dual cofactor usage (NADPH/NADH)

  • The NADPH/NADH preference ratio varies from >100:1 to around 10:1 depending on species

• Inhibition Profiles:

  • Differential sensitivity to product inhibition

  • Varying susceptibility to heavy metals and oxidative damage

These comparative differences may reflect adaptations to different lipid metabolic requirements and environmental conditions across fungal species. Understanding these variations provides insights into the evolution of fatty acid metabolism in fungi and may inform the development of specific inhibitors for pathogenic species .

What is the evolutionary relationship between Emericella nidulans 3-ketoacyl-CoA reductase and similar enzymes in other organisms?

The evolutionary history of 3-ketoacyl-CoA reductase reveals a complex pattern of conservation and divergence across taxonomic groups. Phylogenetic analyses based on sequence homology and structural features provide insights into the evolutionary relationships between the Emericella nidulans enzyme and its counterparts in other organisms.

Evolutionary Classification:

• Emericella nidulans 3-ketoacyl-CoA reductase belongs to the short-chain dehydrogenase/reductase (SDR) superfamily

• Within the SDR superfamily, it clusters with the ketoacyl reductase (KR) family involved in fatty acid biosynthesis

• The enzyme shows the characteristic Rossmann fold for nucleotide binding typical of SDR family proteins

Phylogenetic Relationships:

• Fungal Lineages:

  • Closest homology (85-95% sequence identity) with Aspergillus species

  • Moderate homology (60-75%) with other filamentous ascomycetes (Penicillium, Neurospora)

  • Lower homology (40-55%) with yeasts (Saccharomyces, Candida)

• Cross-Kingdom Comparisons:

  • Substantial homology (35-45%) with plant 3-ketoacyl-CoA reductases

  • Moderate homology (30-40%) with bacterial FabG proteins (bacterial fatty acid biosynthesis)

  • Lower but significant homology (25-35%) with mammalian fatty acid synthase KR domains

Conserved Functional Domains:

• The catalytic triad (Ser, Tyr, Lys) is highly conserved across all lineages

• The NADPH binding motif (TGxxxGxG) shows strong conservation

• Substrate binding regions show greater divergence, reflecting adaptation to different fatty acid profiles

Evolutionary Adaptations:

• Fungal-specific insertions in loop regions that may influence substrate specificity

• Differences in C-terminal regions that could affect protein-protein interactions and localization

• Lineage-specific conservation patterns in residues involved in allosteric regulation

These evolutionary relationships suggest that while the core catalytic mechanism is ancient and conserved, species-specific adaptations have occurred in response to different metabolic requirements. The fungal enzymes appear to have diverged significantly from bacterial and mammalian counterparts, potentially offering opportunities for selective targeting in antifungal drug development .

How can Emericella nidulans 3-ketoacyl-CoA reductase research contribute to understanding fungal pathogenicity and drug development?

Research on Emericella nidulans 3-ketoacyl-CoA reductase provides valuable insights into fungal metabolism that can inform our understanding of fungal pathogenicity and guide antifungal drug development strategies. Although Emericella nidulans itself is not typically considered a primary human pathogen, its close relationship to pathogenic Aspergillus species makes it a valuable model system.

Contributions to Understanding Pathogenicity:

• Metabolic Requirements During Infection:

  • Lipid metabolism is critical for fungal cell membrane integrity and adaptation to host environments

  • 3-Ketoacyl-CoA reductase activity may be upregulated during infection to meet increased demand for membrane lipids

  • Comparative studies between E. nidulans and pathogenic relatives like A. fumigatus can reveal metabolic adaptations that contribute to virulence

• Stress Response Mechanisms:

  • Fatty acid metabolism enzymes, including 3-ketoacyl-CoA reductase, play roles in adapting to environmental stresses encountered during infection

  • Temperature stress, oxidative stress, and nutrient limitation all influence lipid metabolism and membrane composition

• Secondary Metabolite Production:

  • Links between primary metabolism (including fatty acid synthesis) and production of mycotoxins and other virulence factors

  • Some Emericella strains produce sterigmatocystin, a precursor to aflatoxins, which has implications for both human health and agricultural concerns

Applications in Drug Development:

• Target Validation Approach:

  • Genetic knockout studies of AN5861 can establish the essentiality of this enzyme for fungal viability

  • Conditional expression systems can evaluate the vulnerability of this metabolic pathway during different growth phases

• Inhibitor Development Strategy:

  • Structure-based drug design targeting unique features of fungal 3-ketoacyl-CoA reductases

  • High-throughput screening assays using recombinant enzyme to identify selective inhibitors

  • Computational approaches to identify potential binding pockets unique to fungal enzymes

• Potential Therapeutic Advantages:

  • Targeting fatty acid metabolism may affect multiple cellular processes simultaneously

  • Lower potential for resistance development compared to single-target antifungals

  • Possibility for synergistic effects when combined with existing antifungal drugs

Table 3: Potential Therapeutic Applications Based on 3-Ketoacyl-CoA Reductase Research

Understanding the role of 3-ketoacyl-CoA reductase in fungal metabolism provides a foundation for developing novel approaches to combat fungal infections, which remain a significant global health challenge, particularly in immunocompromised populations .

What are the recommended molecular biology techniques for studying Emericella nidulans 3-ketoacyl-CoA reductase expression and regulation?

For comprehensive investigation of Emericella nidulans 3-ketoacyl-CoA reductase expression and regulation, researchers should employ a multi-faceted approach combining genomic, transcriptomic, and proteomic techniques. The following methodological approaches are recommended for different research objectives:

Gene Expression Analysis:

• Quantitative Real-Time PCR (qRT-PCR):

  • Design primers specific to AN5861 gene, avoiding cross-reactivity with related genes

  • Normalize expression using stable reference genes (β-tubulin, actin, or 18S rRNA)

  • Protocol adaptations: RNA extraction using TRIzol with additional purification steps to remove fungal polysaccharides

• RNA-Seq Analysis:

  • Compare transcriptomes under different growth conditions (carbon sources, temperature, pH)

  • Identify co-regulated genes in the fatty acid metabolism pathway

  • Determine alternative splicing patterns if present

• Promoter Analysis:

  • Use 5' RACE to precisely map transcription start sites

  • Clone promoter regions into reporter constructs (GFP, luciferase) for functional analysis

  • Perform chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the promoter

Genetic Manipulation Techniques:

• Gene Disruption Strategies:

  • CRISPR-Cas9 system adapted for filamentous fungi

  • Homologous recombination-based gene replacement

  • Inducible promoter systems for conditional expression

• Protein Tagging Approaches:

  • C-terminal or N-terminal tagging with fluorescent proteins for localization studies

  • Epitope tagging (HA, FLAG, Myc) for immunoprecipitation and protein interaction studies

  • Split-GFP system for protein-protein interaction validation in vivo

Protein Expression and Activity Measurement:

• Western Blot Analysis:

  • Generate specific antibodies against Emericella nidulans 3-ketoacyl-CoA reductase

  • Use phospho-specific antibodies to detect potential regulatory modifications

  • Fractionate cellular components to determine subcellular localization

• Activity-Based Protein Profiling:

  • Design activity-based probes specific for 3-ketoacyl-CoA reductase

  • Use click chemistry to label active enzyme in complex mixtures

  • Combine with mass spectrometry for comprehensive profiling

These molecular approaches can be integrated to develop a comprehensive understanding of how Emericella nidulans regulates 3-ketoacyl-CoA reductase expression in response to environmental conditions, developmental stages, and metabolic requirements .

What bioinformatics resources and tools are available for analyzing Emericella nidulans 3-ketoacyl-CoA reductase structure and function?

Researchers investigating Emericella nidulans 3-ketoacyl-CoA reductase can leverage numerous bioinformatics resources and computational tools to analyze the enzyme's structure, function, and evolutionary relationships. The following resources are particularly valuable for comprehensive analysis:

Sequence Analysis Resources:

• Database Resources:

  • UniProt (Q5B0R9): Primary sequence, functional annotations, and cross-references

  • AspGD (Aspergillus Genome Database): Genome context and gene expression data

  • KEGG (Kyoto Encyclopedia of Genes and Genomes): Metabolic pathway mapping

  • FungiDB: Comparative genomic data across fungal species

• Sequence Analysis Tools:

  • BLAST/PSI-BLAST: Identification of homologs across species

  • MUSCLE/Clustal Omega: Multiple sequence alignment for evolutionary analysis

  • MEGA X: Phylogenetic tree construction and molecular evolution analysis

  • ConSurf: Evolutionary conservation analysis to identify functional sites

Structural Analysis Tools:

• Protein Structure Prediction:

  • AlphaFold2/RoseTTAFold: State-of-the-art protein structure prediction

  • I-TASSER: Integrated platform for structure and function prediction

  • SWISS-MODEL: Homology-based structural modeling

  • ModBase: Database of comparative protein structure models

• Structure Analysis:

  • PyMOL/UCSF Chimera: Visualization and analysis of protein structures

  • CASTp: Prediction of binding pockets and active sites

  • ProDy: Analysis of protein dynamics and flexibility

  • FTMap: Computational mapping of binding hot spots

Functional Prediction Tools:

• Enzyme Function Prediction:

  • EFICAz: Enzyme function inference by combined approach

  • COFACTOR: Structure-based function annotation

  • BioZernike: 3D shape-function relationship analysis

  • EnzymeMiner: Screening for catalytically efficient enzyme variants

• Post-translational Modification Prediction:

  • NetPhos: Phosphorylation site prediction

  • GlycoEP: Glycosylation site prediction

  • GPS-SUMO: SUMOylation site prediction

  • DISOPRED: Prediction of disordered regions

Integrated Analysis Pipelines:

• Systems Biology Tools:

  • COBRA Toolbox: Constraint-based reconstruction and analysis of metabolic networks

  • CellDesigner: Graphical modeling of biochemical networks

  • Cytoscape: Network analysis and visualization platform

  • MetaboAnalyst: Comprehensive tool for metabolomics data analysis

These computational resources enable researchers to generate testable hypotheses about structure-function relationships, substrate specificity determinants, and regulatory mechanisms of Emericella nidulans 3-ketoacyl-CoA reductase. Integration of computational predictions with experimental validation approaches leads to more comprehensive understanding of this important metabolic enzyme .

What experimental model systems are most suitable for studying the physiological role of 3-ketoacyl-CoA reductase in Emericella nidulans?

To elucidate the physiological role of 3-ketoacyl-CoA reductase in Emericella nidulans, researchers can employ various experimental model systems ranging from in vitro biochemical approaches to in vivo fungal systems. Each model system offers distinct advantages for addressing specific research questions.

In Vitro Biochemical Systems:

• Purified Recombinant Enzyme:

  • Determination of intrinsic enzymatic properties

  • Substrate preference and kinetic parameters

  • Inhibitor screening and characterization

  • Structure-function relationship studies using site-directed mutagenesis

• Reconstituted Enzyme Systems:

  • Fatty acid synthesis reconstitution with purified components

  • Analysis of enzyme interdependence within metabolic pathways

  • Investigation of protein-protein interactions with pathway partners

Cellular Model Systems:

• Heterologous Expression Systems:

  • Expression in S. cerevisiae to complement KAR mutants

  • Use of bacterial systems for functional complementation studies

  • Mammalian cell expression for subcellular localization studies

• Emericella nidulans Genetic Manipulation:

  • Gene Disruption/Deletion: Complete knockout to establish essentiality

  • Conditional Promoter Systems: Temperature-sensitive or inducible promoters

  • Allelic Replacement: Introduction of specific mutations to study structure-function

  • Reporter Fusion Constructs: Monitoring expression patterns and regulation

Table 4: Comparative Analysis of Experimental Systems for 3-Ketoacyl-CoA Reductase Research

Physiological Analysis Approaches:

• Growth Phenotyping:

  • Comparative growth analysis on different carbon sources

  • Temperature sensitivity profiling

  • Stress response characterization (oxidative, osmotic, cell wall)

• Lipid Profiling:

  • Lipidomic analysis of membrane composition changes

  • Isotope labeling to track fatty acid synthesis and turnover

  • Specialized media to manipulate fatty acid metabolism

• Developmental Studies:

  • Analysis of asexual and sexual development in mutant strains

  • Microscopic examination of hyphal morphology and conidiation

  • Cell wall composition and integrity testing

The most comprehensive understanding will come from integrating multiple experimental approaches, ranging from in vitro biochemistry to in vivo genetics and physiological studies. This multi-faceted approach allows researchers to connect molecular function to cellular physiology and fungal development .

What are the emerging research trends in studies of fungal 3-ketoacyl-CoA reductases and fatty acid metabolism?

Recent advances in molecular biology techniques, systems biology approaches, and structural biology have driven several emerging research trends in the study of fungal 3-ketoacyl-CoA reductases and fatty acid metabolism. These developments are pushing the field in new directions with potential implications for basic science and applied research.

Current Research Trends:

• Multi-Omics Integration:

  • Combined analysis of genomics, transcriptomics, proteomics, and metabolomics data

  • Network modeling of fatty acid metabolism regulation in response to environmental conditions

  • Identification of regulatory hubs that coordinate primary and secondary metabolism

• Structural Biology Advancements:

  • Cryo-EM studies of fatty acid synthase complexes in filamentous fungi

  • Time-resolved crystallography to capture enzyme catalytic intermediates

  • Application of AlphaFold2 and related AI tools to predict protein-protein interactions in metabolic pathways

• Specialized Lipid Functions:

  • Role of unusual fatty acids in fungal cell signaling

  • Lipid modifications as post-translational regulatory mechanisms

  • Membrane lipid composition changes during morphological transitions and pathogenesis

• Synthetic Biology Applications:

  • Engineering fungal fatty acid metabolism for production of biofuels and specialty chemicals

  • Design of minimal synthetic pathways incorporating fungal enzymes

  • Directed evolution of 3-ketoacyl-CoA reductases for novel substrate specificities

Emerging Methodological Approaches:

• Single-Cell Technologies:

  • Single-cell transcriptomics revealing cell-to-cell variability in metabolic enzyme expression

  • Microfluidic platforms for studying metabolic heterogeneity in fungal populations

  • Spatial transcriptomics mapping enzyme expression across fungal colonies

• Advanced Imaging Techniques:

  • Super-resolution microscopy tracking enzyme localization and dynamics

  • Correlative light and electron microscopy linking enzyme location to ultrastructure

  • Förster resonance energy transfer (FRET) biosensors for monitoring enzyme activity in vivo

• Computational Advancements:

  • Machine learning approaches for predicting enzyme function from sequence

  • Molecular dynamics simulations at extended timescales

  • Quantum mechanics/molecular mechanics (QM/MM) studies of reaction mechanisms

These emerging trends reflect a shift toward more integrated, systems-level understanding of fungal metabolism, with 3-ketoacyl-CoA reductase studied not in isolation but as part of complex, interconnected networks that respond dynamically to environmental and developmental cues .

How might research on Emericella nidulans 3-ketoacyl-CoA reductase contribute to biotechnological applications?

Research on Emericella nidulans 3-ketoacyl-CoA reductase holds significant potential for diverse biotechnological applications, ranging from industrial enzyme production to metabolic engineering for valuable compounds. The unique properties of this enzyme and the metabolic versatility of filamentous fungi create opportunities for innovative applications.

Biocatalysis and Green Chemistry:

• Stereoselective Reduction Applications:

  • Production of chiral alcohols for pharmaceutical intermediates

  • Enantioselective reduction of ketones with high specificity

  • Development of immobilized enzyme systems for continuous processes

• Enzyme Engineering for Industrial Conditions:

  • Protein engineering for enhanced thermostability

  • Modification of cofactor preference (NADH vs. NADPH)

  • Optimization of pH tolerance for industrial processing

Metabolic Engineering Applications:

• Designer Lipid Production:

  • Engineering strains for omega-3 fatty acid production

  • Development of fungi producing structured lipids with specific properties

  • Production of medium-chain fatty acids for biofuel applications

• Secondary Metabolite Enhancement:

  • Manipulation of precursor supply for increased production of valuable metabolites

  • Redirection of metabolic flux from primary to secondary metabolism

  • Engineering of regulatory networks connecting lipid metabolism to secondary metabolite biosynthesis

Table 5: Potential Biotechnological Applications of 3-Ketoacyl-CoA Reductase Research

Enabling Technologies:

• Protein Production Systems:

  • Development of optimized expression hosts for industrial enzyme production

  • Secretion system engineering for extracellular enzyme production

  • Scale-up technologies for fungal cultivation

• Process Integration:

  • Enzyme cascade systems combining multiple catalytic steps

  • Whole-cell biocatalysts with optimized redox cofactor regeneration

  • Continuous flow bioreactors for improved productivity

• Computational Tools:

  • In silico protein engineering for custom substrate specificities

  • Metabolic modeling for strain optimization

  • Process simulations for techno-economic assessment

The biotechnological exploitation of Emericella nidulans 3-ketoacyl-CoA reductase and related enzymes represents a promising avenue for sustainable production of diverse valuable compounds. The inherent catalytic power and selectivity of these enzymes, combined with the metabolic versatility of filamentous fungi, create opportunities for innovative green chemistry applications .

What fundamental questions remain unanswered about the structure and function of Emericella nidulans 3-ketoacyl-CoA reductase?

Despite advances in our understanding of Emericella nidulans 3-ketoacyl-CoA reductase, several fundamental questions remain unanswered. These knowledge gaps represent important research opportunities that could lead to significant advances in fungal metabolism, enzyme catalysis, and biotechnological applications.

Structural Biology Questions:

• Catalytic Mechanism Details:

  • What is the precise arrangement of active site residues during catalysis?

  • How does substrate binding induce conformational changes?

  • What is the rate-limiting step in the catalytic cycle?

• Structural Dynamics:

  • How do protein dynamics contribute to substrate recognition and catalysis?

  • What conformational changes occur during cofactor binding and release?

  • Are there allosteric sites that regulate enzyme activity?

• Protein-Protein Interactions:

  • Does the enzyme function as part of a multi-enzyme complex in vivo?

  • Are there specific protein-protein interactions that regulate activity?

  • How does the enzyme interact with membrane structures in the cell?

Physiological and Regulatory Questions:

• Metabolic Integration:

  • How is 3-ketoacyl-CoA reductase activity coordinated with other enzymes in fatty acid metabolism?

  • What regulatory mechanisms control enzyme expression and activity in response to environmental conditions?

  • How does post-translational modification affect enzyme function in vivo?

• Developmental Regulation:

  • How does 3-ketoacyl-CoA reductase activity change during different developmental stages?

  • Is there differential expression or regulation during sexual and asexual reproduction?

  • What is the role of this enzyme during stress responses and adaptation?

• Evolutionary Aspects:

  • What selective pressures have shaped the evolution of fungal 3-ketoacyl-CoA reductases?

  • How has substrate specificity evolved across fungal lineages?

  • What is the evolutionary history of regulatory mechanisms controlling this enzyme?

Technical Challenges and Future Directions:

• Methodological Innovations Needed:

  • Development of specific inhibitors as chemical probes

  • Real-time assays for monitoring activity in living cells

  • Improved crystallization methods for high-resolution structures

• Integrated Research Approaches:

  • Combining structural biology with systems biology perspectives

  • Linking molecular mechanisms to cellular phenotypes

  • Translating basic science findings to applied biotechnology

Addressing these fundamental questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, systems biology, and evolutionary analysis. The answers will not only advance our basic understanding of fungal metabolism but may also reveal new strategies for antifungal drug development and biotechnological applications .