Recombinant Candida albicans 3-ketoacyl-CoA reductase (CaO19.11340, CaO19.3859)

What is Candida albicans 3-ketoacyl-CoA reductase and what is its biological function?

3-ketoacyl-CoA reductase (also known as 3-ketoreductase or KAR, EC 1.1.1.-) is an important enzyme in the fatty acid metabolism pathway of Candida albicans. This enzyme catalyzes the second step in the fatty acid elongation (FAE) cycle, specifically the reduction of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA using NADPH as a cofactor . This reaction is critical for the biosynthesis of very long chain fatty acids (VLCFAs), which serve as building blocks for diverse lipids in eukaryotic organisms.

The full amino acid sequence of the C. albicans 3-ketoacyl-CoA reductase consists of 349 amino acids and is characterized as a microsomal beta-keto-reductase . The protein contains specific domains that enable its enzymatic activity, including substrate binding regions and cofactor interaction sites necessary for the reduction reaction.

How does C. albicans 3-ketoacyl-CoA reductase compare to similar enzymes in other organisms?

While specific comparative data for 3-ketoacyl-CoA reductase across multiple species is limited in the search results, we can draw some parallels from related enzymes. Unlike the 3-ketoacyl-CoA thiolases, where C. albicans expresses three distinct enzymes (Pot1p, Fox3p, and Pot13p) compared to only one in Saccharomyces cerevisiae, the evolutionary distribution of 3-ketoacyl-CoA reductase shows variations in substrate specificity and cellular localization .

In contrast to bacterial systems like Pseudomonas aeruginosa, where the NADPH-dependent FabG (3-ketoacyl reductase) has been demonstrated to use CoA esters as substrates and participate in poly-3-hydroxyalkanoate (PHA) synthesis, the C. albicans enzyme functions primarily in the fatty acid elongation pathway . While bacterial FabG typically works with acyl carrier protein (ACP)-linked substrates in fatty acid synthesis, it can also process CoA-linked substrates up to C18 in chain length .

What is the role of 3-ketoacyl-CoA reductase in the fatty acid elongation cycle?

The fatty acid elongation (FAE) cycle consists of four sequential reactions that cumulatively extend acyl chains by two carbon atoms per cycle. The process follows this order:

• Condensation: A Claisen condensation between an acyl-CoA and malonyl-CoA, catalyzed by either ELONGATION DEFECTIVE LIKE (ELO) or 3-ketoacyl-CoA synthase (KCS) enzymes, generating a 3-ketoacyl-CoA intermediate

• Reduction: The 3-ketoacyl-CoA is reduced to 3-hydroxyacyl-CoA by 3-ketoacyl-CoA reductase (KCR)

• Dehydration: The 3-hydroxyacyl-CoA is dehydrated to trans-2-enoyl-CoA by 3-hydroxyacyl-CoA dehydratase (HCD)

• Reduction: The final step involves reduction of the enoyl-CoA to an acyl-CoA by enoyl-CoA reductase (ECR)

This complete cycle results in an acyl-CoA product that is two carbon atoms longer than the initial substrate, which can then enter the next round of elongation or be utilized for various cellular processes.

What are the optimal conditions for expressing and purifying recombinant C. albicans 3-ketoacyl-CoA reductase?

Based on available information for recombinant C. albicans 3-ketoacyl-CoA reductase production, the following conditions are recommended:

Expression System:
While specific expression conditions for C. albicans 3-ketoacyl-CoA reductase are not detailed in the search results, heterologous expression in Escherichia coli has proven successful for related enzymes from C. albicans, such as dihydroorotate dehydrogenase . Both full-length and N-terminally truncated versions (lacking targeting sequences and transmembrane domains) have been successfully expressed in bacterial systems.

Purification and Storage:

• Buffer composition: Tris-based buffer with 50% glycerol optimized for protein stability

• Storage conditions: Store at -20°C for regular use; -80°C for extended storage

• Working aliquots: Maintain at 4°C for up to one week

• Stability note: Repeated freezing and thawing is not recommended as it may compromise enzymatic activity

What assay methods can be used to measure 3-ketoacyl-CoA reductase activity?

While the search results don't provide a specific protocol for C. albicans 3-ketoacyl-CoA reductase activity measurement, activity assays for related enzymes can be adapted. A standard approach would involve:

• Substrate preparation: Utilize 3-ketoacyl-CoA substrates of varying chain lengths

• Cofactor addition: Include NADPH as the electron donor

• Activity measurement: Monitor NADPH oxidation at 340 nm spectrophotometrically

• Quantification: Calculate activity in mU/mg protein (typical values for recombinant ketoacyl-CoA reductase expression range from 50-90 mU/mg total protein compared to background levels of 10-20 mU/mg in control samples)

For reference, when assaying ketoacyl-CoA reductase activity in recombinant systems, proper controls must be established to account for endogenous background activity. In E. coli expression systems, vector-only controls typically show background activity of approximately 10-20 mU/mg protein, likely due to native E. coli FabG activity .

How can researchers overcome common challenges in working with recombinant 3-ketoacyl-CoA reductase?

Several experimental challenges may arise when working with 3-ketoacyl-CoA reductase:

Protein Stability Issues:

• Include 50% glycerol in storage buffers to prevent activity loss

• Maintain small working aliquots at 4°C rather than repeatedly freeze-thawing samples

• Consider the addition of reducing agents if the enzyme contains critical cysteine residues

Expression Optimization:

• When expressing in heterologous systems, codon optimization for the host organism may improve yield

• For membrane-associated enzymes, consider expressing truncated versions lacking transmembrane domains, as demonstrated with C. albicans dihydroorotate dehydrogenase

Activity Verification:

• Include appropriate positive and negative controls in enzyme assays

• Account for background activity from host enzymes (typically 10-20 mU/mg protein in E. coli systems)

• Verify substrate specificity using multiple chain-length substrates

How might 3-ketoacyl-CoA reductase contribute to C. albicans pathogenicity and virulence?

Investigations of 3-ketoacyl-CoA thiolases in C. albicans revealed that β-oxidation appears to be dispensable for virulence. When single, double, and triple mutants of Pot1p, Fox3p, and Pot13p were assessed in an embryonated chicken egg infection model, no significant attenuation was observed, confirming previous assumptions about the nonessential nature of β-oxidation for C. albicans virulence .

How can 3-ketoacyl-CoA reductase be targeted for antifungal drug development?

While the search results don't directly address inhibitors of C. albicans 3-ketoacyl-CoA reductase, lessons can be drawn from inhibitor screening approaches used for other C. albicans enzymes:

For C. albicans dihydroorotate dehydrogenase (DHODH), researchers conducted an inhibitor screening with 28 selected compounds. Only specific compounds, including a dianisidine derivative (redoxal) and a biphenyl quinoline-carboxylic acid derivative (brequinar sodium), showed significant inhibition. Notably, these were known potent inhibitors of mammalian DHODH, suggesting conservation of inhibitor sensitivity across species .

A similar approach could be applied to 3-ketoacyl-CoA reductase:

• Conduct comparative structural analysis between fungal and human enzymes to identify unique features

• Screen known inhibitors of related enzymes across different species

• Develop selective inhibitors that target fungal-specific structural elements

• Assess effects on fungal growth, morphology, and virulence

Importantly, when considering 3-ketoacyl-CoA reductase as a drug target, researchers must account for the finding that some fatty acid metabolism pathways appear dispensable for virulence, which might limit therapeutic efficacy .

What phenotypic effects would be expected from 3-ketoacyl-CoA reductase gene mutations in C. albicans?

While direct phenotypic data for 3-ketoacyl-CoA reductase mutations in C. albicans is not provided in the search results, insights can be drawn from studies on related enzymes in the fatty acid metabolism pathway.

In the case of 3-ketoacyl-CoA thiolases (Pot1p, Fox3p, and Pot13p), phenotypic characterization of single, double, and triple mutants revealed distinct functional contributions:

These findings demonstrate functional specialization among related enzymes, with some being essential for specific metabolic processes while others play more redundant roles .

For 3-ketoacyl-CoA reductase, potential phenotypic effects of mutations might include:

• Altered fatty acid elongation patterns

• Changes in membrane lipid composition

• Potential growth defects under specific environmental conditions

• Possible alterations in stress response or adaptation to host environments

How can contradictory or variable results in 3-ketoacyl-CoA reductase studies be reconciled?

When encountering contradictory results in 3-ketoacyl-CoA reductase studies, researchers should consider several factors that might contribute to experimental variability:

• Background enzyme activity: Control strains can exhibit baseline reductase activity (10-20 mU/mg protein), potentially masking small but significant changes in experimental conditions

• Enzymatic redundancy: Related enzymes may partially compensate for 3-ketoacyl-CoA reductase deficiency, as observed with 3-ketoacyl-CoA thiolases where multiple enzymes showed overlapping functions

• Experimental conditions: Temperature, pH, substrate concentration, and cofactor availability can significantly impact enzyme activity measurements

• Strain variations: Different C. albicans strains may show variable enzyme expression or activity levels

To reconcile contradictory results, researchers should:

• Utilize appropriate statistical analyses across multiple independent experiments

• Implement consistent experimental protocols with well-defined controls

• Consider environmental variables that might influence enzyme activity

• Validate findings using complementary approaches (genetic, biochemical, and structural)

What controls and standards should be included in 3-ketoacyl-CoA reductase research protocols?

Proper experimental design for 3-ketoacyl-CoA reductase studies should include the following controls and standards:

For Enzyme Activity Assays:

• Negative controls: Vector-only or empty vector transformants to establish background activity (typically 10-20 mU/mg protein in E. coli systems)

• Positive controls: Well-characterized related enzymes with established activity profiles

• Substrate controls: Varying chain-length substrates to assess specificity

• Cofactor controls: Reactions with and without NADPH to confirm cofactor dependency

For Gene Function Studies:

• Wild-type reference strains

• Gene deletion mutants

• Complemented mutants to confirm phenotype rescue

• Multiple independent transformants to account for clonal variations

For Protein Expression and Purification:

• SDS-PAGE analysis of purified protein with molecular weight standards

• Western blot confirmation of protein identity

• Activity assays of purified enzyme compared to crude extracts

• Storage stability controls (activity measured over time under different storage conditions)

How might comparative genomics inform our understanding of 3-ketoacyl-CoA reductase evolution and function?

Comparative genomic approaches could provide valuable insights into the evolution and functional specialization of 3-ketoacyl-CoA reductase across fungal species. Similar to the phylogenetic analysis conducted for 3-ketoacyl-CoA thiolases, where C. albicans possesses three enzymes compared to a single enzyme in S. cerevisiae, such analyses could reveal:

• Evolutionary relationships between 3-ketoacyl-CoA reductases across fungal species

• Potential gene duplication events and subsequent functional divergence

• Correlation between enzyme diversity and species-specific metabolic adaptations

• Conservation of catalytic domains versus divergence in regulatory regions

This approach could reveal whether the presence of specific 3-ketoacyl-CoA reductase variants correlates with pathogenicity, host range, or environmental adaptation across fungal species .

What techniques can be applied to study the interaction of 3-ketoacyl-CoA reductase with other components of the fatty acid elongation complex?

3-ketoacyl-CoA reductase functions as part of the multi-component fatty acid elongase complex, where its activity is coordinated with other enzymes in the pathway . Several techniques could be employed to study these protein-protein interactions:

• Co-immunoprecipitation: Using antibodies against 3-ketoacyl-CoA reductase to pull down associated proteins from C. albicans lysates

• Yeast two-hybrid screening: Identifying direct protein interactions between 3-ketoacyl-CoA reductase and other fatty acid metabolism enzymes

• Bimolecular fluorescence complementation (BiFC): Visualizing protein interactions in vivo by tagging potential interacting partners with complementary fluorescent protein fragments

• Cross-linking mass spectrometry: Identifying interaction interfaces between 3-ketoacyl-CoA reductase and partner proteins

• Cryo-electron microscopy: Resolving the structure of the entire fatty acid elongase complex to understand spatial relationships between component enzymes

How might systems biology approaches enhance our understanding of 3-ketoacyl-CoA reductase in the context of C. albicans metabolism?

Systems biology approaches could provide a comprehensive understanding of 3-ketoacyl-CoA reductase within the broader context of C. albicans metabolism, particularly considering the finding that some fatty acid metabolism pathways appear dispensable for virulence . These approaches might include:

• Metabolomic profiling: Comparing wild-type and 3-ketoacyl-CoA reductase mutant strains to identify changes in fatty acid profiles and related metabolites

• Transcriptomic analysis: Examining gene expression changes in response to 3-ketoacyl-CoA reductase perturbation, revealing compensatory pathways

• Flux analysis: Quantifying changes in metabolic flux through fatty acid biosynthesis and related pathways

• Mathematical modeling: Developing predictive models of fatty acid metabolism that incorporate 3-ketoacyl-CoA reductase activity and regulation

• Multi-omics integration: Combining genomic, transcriptomic, proteomic, and metabolomic data to build comprehensive models of fatty acid metabolism in C. albicans under different conditions

Such approaches could reveal how 3-ketoacyl-CoA reductase activity influences broader cellular processes, including membrane composition, stress response, morphogenesis, and host-pathogen interactions.