Recombinant Coccidioides immitis 3-ketoacyl-CoA reductase (CIMG_08188)

What is 3-ketoacyl-CoA reductase in Coccidioides immitis and what is its biological function?

3-ketoacyl-CoA reductase (KAR, EC 1.1.1.-) in Coccidioides immitis, encoded by the CIMG_08188 gene, is an essential enzyme involved in fatty acid biosynthesis pathways. It belongs to the short-chain dehydrogenase/reductase (SDR) family and catalyzes the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor in the elongation cycle of fatty acid synthesis.

The enzyme plays a crucial role in the fungal cell membrane and cell wall integrity by contributing to the production of long-chain fatty acids and complex lipids. The protein consists of 349 amino acids and contains characteristic NAD(P)-binding motifs and catalytic residues typical of the SDR family .

This enzyme is also known by alternative names such as 3-ketoreductase (KAR) and microsomal beta-keto-reductase, reflecting its functional role in reducing keto groups during fatty acid synthesis . The importance of this enzyme in fungal metabolism makes it a potential target for antifungal drug development.

What are the optimal laboratory conditions for handling recombinant Coccidioides immitis 3-ketoacyl-CoA reductase?

For optimal handling of recombinant Coccidioides immitis 3-ketoacyl-CoA reductase in laboratory settings, the following conditions should be considered:

Storage Conditions:

• Store the purified protein at -20°C for regular use

• For extended storage, maintain at -20°C to -80°C

• Store in Tris-based buffer with 50% glycerol as a stabilizing agent

• Avoid repeated freeze-thaw cycles which can compromise enzyme activity

Working Conditions:

• For active experiments, maintain working aliquots at 4°C for up to one week

• Perform enzymatic assays at pH 7.0-7.5 in appropriate buffer systems

• Include reducing agents (e.g., DTT or β-mercaptoethanol) at low concentrations to maintain the integrity of cysteine residues

Activity Assessment:

• Monitor enzyme activity using spectrophotometric assays measuring NADPH oxidation at 340 nm

• Maintain temperature control at 25-30°C during activity assays to reflect the physiological conditions of the fungal pathogen

Following these guidelines will help ensure the maintenance of protein stability and functional integrity throughout experimental procedures. This methodical approach prevents data variability stemming from protein degradation or activity loss.

How can researchers express and purify recombinant Coccidioides immitis 3-ketoacyl-CoA reductase?

The expression and purification of recombinant Coccidioides immitis 3-ketoacyl-CoA reductase requires a systematic approach:

Expression System Selection:

Expression Methodology:

• Clone the CIMG_08188 gene into an appropriate expression vector with a suitable purification tag (His6, GST, etc.)

• Transform into the chosen expression system

• Optimize expression conditions (temperature, induction timing, media composition)

• For E. coli expression, consider using lower temperatures (16-20°C) and longer induction times to improve solubility

Purification Protocol:

• Lyse cells in appropriate buffer (typically Tris-based with protease inhibitors)

• Clarify lysate by centrifugation (15,000×g, 30 min, 4°C)

• For His-tagged protein:
  a. Apply clarified lysate to Ni-NTA resin
  b. Wash with increasing imidazole concentrations (20-40 mM)
  c. Elute with high imidazole (250-300 mM)

• Perform buffer exchange to remove imidazole

• Consider secondary purification step (ion exchange or size exclusion chromatography)

• Store in stabilizing buffer containing 50% glycerol

Quality Control:

• Assess purity by SDS-PAGE (>90% purity desired)

• Verify identity by mass spectrometry

• Test activity using appropriate enzymatic assays

• Optimize storage conditions to maintain stability

This comprehensive approach ensures the production of high-quality recombinant enzyme suitable for downstream applications in structural and functional studies.

What is the role of 3-ketoacyl-CoA reductase in the metabolic pathways of Coccidioides immitis?

3-ketoacyl-CoA reductase plays a pivotal role in several metabolic pathways in Coccidioides immitis:

Fatty Acid Biosynthesis:
The enzyme catalyzes a crucial reduction step in the fatty acid elongation cycle, converting 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor. This reaction is part of the iterative process that adds two-carbon units to growing fatty acid chains, essential for membrane lipid biosynthesis.

Cell Wall Biosynthesis:
The fatty acids produced through pathways involving 3-ketoacyl-CoA reductase contribute to the synthesis of complex lipids that form part of the fungal cell wall. These components are crucial for cell integrity and resistance to environmental stresses.

Virulence and Pathogenicity:
Proper cell wall and membrane composition are essential for fungal pathogenicity. The lipids produced through pathways involving 3-ketoacyl-CoA reductase likely contribute to:

• Host-pathogen interactions

• Resistance to host defense mechanisms

• Adaptation to the host environment during infection

Integration with Other Metabolic Networks:
3-ketoacyl-CoA reductase functions within a complex metabolic network that connects with:

• Carbohydrate metabolism (providing precursors)

• Energy metabolism (using NADPH generated from the pentose phosphate pathway)

• Secondary metabolite production (using fatty acid-derived precursors)

The enzyme's position at this metabolic crossroad makes it not only essential for basic cellular functions but also potentially important for the unique morphological changes that occur during the parasitic cycle of Coccidioides, possibly similar to the role of chitinases in endospore differentiation described in other research .

Understanding these metabolic interconnections provides valuable insights for researchers developing targeted approaches to disrupt fungal metabolism for therapeutic purposes.

How does the structure of Coccidioides immitis 3-ketoacyl-CoA reductase compare to homologous enzymes in other pathogenic fungi?

Comparative structural analysis of Coccidioides immitis 3-ketoacyl-CoA reductase with homologous enzymes from other pathogenic fungi reveals important evolutionary and functional relationships:

Structural Conservation:
Sequence alignment and homology modeling indicate that the core catalytic domain and the Rossmann fold for NAD(P)H binding are highly conserved across fungal species. The Coccidioides immitis enzyme shares approximately 45-60% sequence identity with orthologs from Aspergillus, Candida, and Cryptococcus species.

Key Differences:

Phylogenetic Analysis:
Phylogenetic analysis places the C. immitis 3-ketoacyl-CoA reductase in a distinct clade among soil-dwelling dimorphic fungi, separate from the clusters formed by yeasts (Candida, Saccharomyces) and molds (Aspergillus). This evolutionary divergence may reflect adaptation to the unique lifecycle of Coccidioides, which includes both saprophytic and parasitic phases.

Functional Implications:
The structural differences observed in the substrate-binding pocket suggest that C. immitis 3-ketoacyl-CoA reductase may have evolved specific substrate preferences optimized for the unique metabolic requirements of its parasitic lifecycle. These differences can be exploited for the development of species-specific inhibitors with potential antifungal applications.

Methodological Approaches for Further Investigation:

• X-ray crystallography or cryo-EM studies to determine the actual structure

• Molecular dynamics simulations to identify flexible regions and binding pocket dynamics

• Site-directed mutagenesis of non-conserved residues to assess their role in substrate specificity

• Enzyme kinetics with various substrates to quantify functional differences

This comparative structural analysis provides a foundation for understanding the specialized function of C. immitis 3-ketoacyl-CoA reductase and its potential as a target for selective therapeutic intervention.

What approaches can be used to design selective inhibitors targeting 3-ketoacyl-CoA reductase for antifungal development?

Designing selective inhibitors targeting Coccidioides immitis 3-ketoacyl-CoA reductase for antifungal development requires a sophisticated multi-disciplinary approach:

Structure-Based Drug Design Strategies:

• Virtual Screening Approach:

  • Generate a high-quality homology model of C. immitis 3-ketoacyl-CoA reductase based on crystal structures of homologous enzymes

  • Identify unique binding pockets in the C. immitis enzyme not present in human orthologs

  • Perform virtual screening of compound libraries using molecular docking

  • Prioritize compounds that show selective binding to fungal over human enzymes

• Fragment-Based Design:

  • Identify small molecular fragments that bind to different regions of the active site

  • Link compatible fragments to create high-affinity, selective inhibitors

  • Optimize these compounds through iterative medicinal chemistry

Rational Inhibitor Design Based on Catalytic Mechanism:

3-ketoacyl-CoA reductase follows an ordered Bi-Bi mechanism where NADPH binds first, followed by the ketoacyl-CoA substrate. Potential inhibition strategies include:

Experimental Validation Pipeline:

• In vitro Screening:

  • Develop a high-throughput enzymatic assay monitoring NADPH oxidation

  • Screen candidate compounds for inhibitory activity (IC50 determination)

  • Determine mechanism of inhibition through detailed kinetic studies

  • Test selectivity against human orthologs and other essential fungal enzymes

• Cellular Assays:

  • Evaluate antifungal activity against C. immitis in culture

  • Assess impact on fatty acid composition using lipidomics

  • Determine effects on cell wall integrity and morphology

  • Evaluate cytotoxicity against mammalian cells

• Structural Confirmation:

  • Use X-ray crystallography to confirm binding mode of promising inhibitors

  • Refine inhibitor structure based on observed interactions

This systematic approach leverages the unique structural features of C. immitis 3-ketoacyl-CoA reductase to develop selective inhibitors with potential application as novel antifungal agents against coccidioidomycosis.

How can gene editing techniques be applied to study the function of CIMG_08188 in Coccidioides immitis?

Gene editing techniques offer powerful approaches to elucidate the function of CIMG_08188 in Coccidioides immitis, though their application requires careful consideration of biosafety due to the pathogenic nature of this organism:

CRISPR-Cas9 System for CIMG_08188 Modification:

• Design Strategy:

  • Design sgRNAs targeting specific regions of CIMG_08188

  • Create repair templates for:
    a) Complete gene knockout
    b) Point mutations in catalytic residues
    c) Domain-specific deletions
    d) Promoter modifications for controlled expression

• Optimized Delivery Protocol:

  • Transform protoplasts with ribonucleoprotein (RNP) complexes containing:
    a) Recombinant Cas9 protein
    b) In vitro transcribed sgRNA
    c) Repair template DNA

  • Select transformants using appropriate markers

• Validation of Edits:

  • PCR amplification and sequencing of the target locus

  • Western blotting to confirm protein expression changes

  • Enzyme activity assays to confirm functional alteration

Alternative Approaches for Genetic Manipulation:

Functional Analysis of Modified Strains:

• Phenotypic Characterization:

  • Growth rates under various conditions

  • Morphological changes during both saprophytic and parasitic phases

  • Cell wall integrity assays

  • Lipid composition analysis

  • Virulence assessment in appropriate models

• Molecular Phenotyping:

  • Transcriptomics to identify compensatory pathways

  • Proteomics to assess global protein changes

  • Metabolomics focusing on fatty acid and lipid profiles

  • Flux analysis to determine metabolic rewiring

• Complementation Studies:

  • Reintroduction of wild-type CIMG_08188

  • Introduction of CIMG_08188 variants with specific mutations

  • Expression of orthologs from other species

  • Domain swapping with related enzymes

The approach described here draws upon strategies similar to those used for chitinase gene disruption in Coccidioides, where targeted gene modifications led to significant insights into fungal biology and pathogenesis . Similar methodologies could be applied to study CIMG_08188, with modifications accounting for the specific challenges associated with this gene and its encoded enzyme.

What are the implications of 3-ketoacyl-CoA reductase in virulence and vaccine development for Coccidioides immitis?

The implications of 3-ketoacyl-CoA reductase in virulence and vaccine development for Coccidioides immitis represent a frontier in coccidioidomycosis research:

Role in Fungal Virulence:

3-ketoacyl-CoA reductase likely contributes to virulence through several mechanisms:

• Cell Wall Integrity:
  The enzyme's role in fatty acid biosynthesis directly impacts cell wall composition and integrity, which are essential for fungal survival within host environments.

• Adaptation to Host Environment:
  Fatty acid metabolism modifications may help the fungus adapt to the nutrient-limited environment inside host cells, similar to how other metabolic enzymes contribute to pathogen adaptation.

• Resistance to Host Defenses:
  Modified cell membrane composition can provide resistance to host antimicrobial peptides and oxidative stress.

• Morphological Transitions:
  The enzyme may be involved in the lipid remodeling necessary for the spherule-endospore transition, a critical process for Coccidioides pathogenesis.

Potential in Vaccine Development:

Evidence from Related Research:

Research on genetically engineered live attenuated vaccines for Coccidioides provides valuable insights. Similar to the approach used with chitinase genes (CTS2 and CTS3) , modifications to CIMG_08188 could potentially create attenuated strains with reduced virulence but retained immunogenicity.

The success of chitinase-deficient strains in protecting both BALB/c and C57BL/6 mice against coccidioidomycosis suggests that targeting key metabolic enzymes can be a viable strategy for vaccine development. These attenuated strains induced immune responses characterized by both T-helper-1 and T-helper-2-type cytokines, which are essential for effective protection against fungal infections.

Future Research Directions:

• Evaluate the impact of CIMG_08188 deletion or modification on Coccidioides virulence

• Assess the immunogenicity of recombinant 3-ketoacyl-CoA reductase

• Investigate attenuated strains with modified CIMG_08188 as potential vaccine candidates

• Identify immunodominant epitopes within the enzyme for epitope-based vaccine design

These approaches could lead to novel strategies for preventing coccidioidomycosis, a disease that causes significant morbidity in endemic regions.

How can enzyme kinetics and inhibition studies of 3-ketoacyl-CoA reductase inform drug development strategies?

Enzyme kinetics and inhibition studies of Coccidioides immitis 3-ketoacyl-CoA reductase provide crucial information for rational drug development strategies:

Comprehensive Kinetic Analysis:

• Steady-State Kinetics:

  • Determine Km and kcat values for various chain-length substrates

  • Establish the preferred substrate profile

  • Define the cofactor preference (NADH vs. NADPH) and associated kinetic parameters

• Reaction Mechanism:

  $E + NADPH \rightleftharpoons E \cdot NADPH + 3-ketoacyl-CoA \rightleftharpoons E \cdot NADPH \cdot 3-ketoacyl-CoA \rightarrow E \cdot NADP^+ \cdot 3-hydroxyacyl-CoA \rightleftharpoons E \cdot NADP^+ + 3-hydroxyacyl-CoA \rightleftharpoons E + NADP^+$

  • Determine if the mechanism follows ordered Bi Bi kinetics (typical for dehydrogenases/reductases)

  • Identify rate-limiting steps through pre-steady-state kinetics

  • Map transition states that could be targeted by inhibitors

Advanced Inhibition Studies:

Translation to Drug Development:

• Target Product Profile Development:

  • Define required potency (IC50 < 100 nM)

  • Establish selectivity requirements (>100-fold vs. human ortholog)

  • Determine physicochemical parameters (cLogP, MW, PSA) for antifungal efficacy

• Hit-to-Lead Optimization Strategy:

  • Use enzyme-inhibitor complex structures to guide medicinal chemistry

  • Optimize residence time rather than focusing solely on equilibrium constants

  • Consider allosteric inhibition to achieve greater selectivity

• Integrated Approach:

  • Correlate enzyme inhibition with whole-cell antifungal activity

  • Establish pharmacokinetic/pharmacodynamic (PK/PD) relationships

  • Develop combination strategies with existing antifungals

Case Study Design for Novel Inhibitor Development:

A systematic approach for developing C. immitis 3-ketoacyl-CoA reductase inhibitors would include:

• Initial screening of focused libraries based on known SDR inhibitors

• Detailed kinetic characterization of hits to determine inhibition mechanism

• Co-crystallization with promising inhibitors

• Structure-guided optimization cycles

• Evaluation of optimized compounds in cellular and in vivo models

This comprehensive approach to enzyme kinetics and inhibition studies provides a strong foundation for developing selective inhibitors of C. immitis 3-ketoacyl-CoA reductase with potential as novel antifungal agents.

What analytical techniques are most effective for studying the structure-function relationships of recombinant Coccidioides immitis 3-ketoacyl-CoA reductase?

Multiple analytical techniques can be employed in a complementary manner to elucidate the structure-function relationships of recombinant Coccidioides immitis 3-ketoacyl-CoA reductase:

Structural Analysis Techniques:

• X-ray Crystallography:

  • Provides atomic-level resolution of protein structure

  • Enables visualization of:

    • Cofactor binding pocket

    • Substrate binding site

    • Catalytic residues orientation

  • Co-crystallization with substrates, products, or inhibitors reveals binding modes

  • Resolution target: 1.5-2.5 Å for detailed catalytic mechanism insights

• Cryo-Electron Microscopy:

  • Alternative for structure determination if crystallization proves challenging

  • Can capture different conformational states

  • Particularly useful if the enzyme forms larger complexes with other proteins

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Analyzes protein dynamics in solution

  • Identifies flexible regions involved in catalysis

  • Maps chemical shift perturbations upon ligand binding

  • Best for studying localized regions rather than the entire 349-amino acid protein

Functional Analysis Approaches:

Structure-Function Correlation Methods:

• Site-Directed Mutagenesis:

  • Systematic replacement of putative catalytic residues

  • Creation of chimeric enzymes with related reductases

  • Domain swapping experiments

  • Alanine scanning of substrate binding pocket

• Molecular Dynamics Simulations:

  • Models protein flexibility and conformational changes

  • Simulates enzyme-substrate interactions in atomic detail

  • Identifies water networks important for catalysis

  • Predicts effects of mutations on structure and function

Integrated Experimental Workflow:

A comprehensive structure-function analysis would follow this workflow:

• Obtain high-resolution structure through X-ray crystallography or cryo-EM

• Identify putative catalytic and binding residues through structural analysis

• Confirm roles through site-directed mutagenesis and kinetic analysis

• Explore conformational dynamics using NMR and MD simulations

• Map the complete catalytic cycle through pre-steady-state kinetics

• Validate findings by designing structure-based mutations with predictable effects on function

This multi-technique approach provides a comprehensive understanding of how the enzyme's structure relates to its catalytic function, which is essential for both fundamental enzymology research and applied studies targeting this enzyme for drug development.