Recombinant Escherichia fergusonii 3-ketoacyl-CoA thiolase (fadA), partial,Yeast

What distinguishes E. fergusonii 3-ketoacyl-CoA thiolase from related bacterial species?

E. fergusonii 3-ketoacyl-CoA thiolase belongs to the thiolase enzyme family that catalyzes the final step in fatty acid β-oxidation. While E. fergusonii and E. coli share significant genetic homology, molecular identification techniques reveal distinct differences in their thiolase genes. PCR-based detection methods can effectively differentiate E. fergusonii from related species through targeted amplification of specific gene sequences . Research indicates that E. fergusonii frequently carries gene sequences homologous to those found in E. coli, with studies detecting the uidA gene in E. fergusonii strains . This genetic similarity creates challenges for accurate identification using biochemical methods alone, necessitating molecular approaches for definitive characterization.

How does the subcellular localization of thiolases differ between bacteria and yeast systems?

In bacteria like E. fergusonii, thiolases operate in the cytoplasm as part of the fatty acid degradation pathway. In contrast, yeast systems exhibit compartmentalized thiolase distribution:

Unlike bacteria, yeasts like Candida albicans contain multiple thiolases with distinct subcellular targeting. Phylogenetic analysis reveals that C. albicans Pot1p and Fox3p cluster with the single S. cerevisiae peroxisomal thiolase, while Pot13p contains a putative mitochondrial import sequence and clusters with thiolases from filamentous fungi . This compartmentalization reflects fundamental differences in metabolic organization between prokaryotic and eukaryotic systems.

What evolutionary relationships exist between bacterial fadA and yeast thiolases?

Phylogenetic analysis indicates complex evolutionary relationships between bacterial and yeast thiolases. In C. albicans, three distinct 3-ketoacyl-CoA thiolases (Pot1p, Fox3p, and Pot13p) show varying relationships to bacterial homologs . The close clustering of Pot1p and Fox3p suggests a relatively recent gene duplication event, while the more distantly related Pot13p appears to have evolved from an ancient ketoacyl-CoA thiolase precursor .

Phylogenetic distances between these thiolases are quantified in the following table:

This data demonstrates that bacterial thiolases like E. fergusonii fadA likely share common ancestral origins with specific yeast thiolase lineages, with sequence divergence reflecting specialized adaptations to different metabolic requirements and cellular compartmentalization .

What are the key structural domains and catalytic residues in E. fergusonii fadA?

The 3-ketoacyl-CoA thiolase (fadA) from E. fergusonii contains highly conserved domains characteristic of the thiolase enzyme family. The catalytic core includes:

• A nucleophilic cysteine residue that forms a covalent intermediate with the substrate

• Conserved histidine and cysteine residues forming the catalytic triad

• CoA-binding pockets that accommodate substrate positioning

• Dimerization interface domains essential for quaternary structure

Mutagenesis studies demonstrate that specific amino acid residues are critical for proper enzyme function. For example, mutations like L14A and L76A in the related FadA protein can disrupt protein production and structural integrity, as observed in the failure to produce pre-FadA and absence of filamentous structures . These structural insights help researchers understand the molecular basis of enzyme activity and provide targets for functional studies.

How do post-translational modifications affect thiolase function across species?

Post-translational modifications significantly impact thiolase activity and regulation in both bacterial and yeast systems. Key modifications include:

In bacterial systems, the FadA protein undergoes crucial processing from pre-FadA to mature FadA, which affects its functionality. Research demonstrates that pre-FadA represents a key component of amyloid-like FadA structures, highlighting the importance of proteolytic processing in determining enzyme properties .

What expression systems are optimal for producing recombinant E. fergusonii fadA?

Selecting the appropriate expression system is critical for obtaining functional recombinant E. fergusonii fadA. The following methodological approaches have proven effective:

• Bacterial Expression Systems:

  • E. coli BL21(DE3) with pET vectors offers high-yield expression

  • Cold-shock induction (16-18°C) improves solubility of recombinant fadA

  • Co-expression with molecular chaperones (GroEL/GroES) enhances proper folding

• Yeast Expression Systems:

  • Pichia pastoris provides eukaryotic processing capabilities

  • S. cerevisiae pot1Δ mutants allow functional complementation studies

  • C. albicans expression systems enable direct comparison with native thiolases

Expression optimization requires careful consideration of induction conditions, as demonstrated in studies of related thiolases. For instance, in C. albicans, different thiolase family members (Pot1p, Fox3p, Pot13p) require specific expression conditions for optimal activity . These considerations equally apply to recombinant E. fergusonii fadA production.

What assays are most reliable for measuring 3-ketoacyl-CoA thiolase activity?

Several assay methods provide reliable measurements of thiolase activity, each with specific advantages:

When implementing these assays, researchers must carefully control reaction conditions including pH, temperature, and substrate concentrations. For studying E. fergusonii fadA specifically, comparative analysis with yeast thiolases can provide valuable insights into substrate specificity and catalytic efficiency. Research on C. albicans thiolases demonstrates the importance of temperature selection, as different thiolases show optimal activity at different temperatures—a consideration that likely applies to bacterial thiolases as well .

How does substrate preference differ between E. fergusonii fadA and yeast thiolases?

Substrate specificity profiles reveal significant differences between bacterial and yeast thiolases:

Research demonstrates that growth capacity on different fatty acids varies significantly between fungi and bacteria. While C. albicans cannot utilize fatty acids with a chain length of 8-12 carbon atoms, it can efficiently metabolize longer-chain fatty acids . This contrasts with filamentous fungi like A. fumigatus, which can utilize a broader range of fatty acids including C12 but not C8-C10 . These differences likely reflect adaptations to different ecological niches and metabolic requirements.

How do temperature and pH affect the catalytic efficiency of thiolases across species?

Temperature and pH significantly impact thiolase activity in both bacterial and yeast systems:

Experimental evidence indicates that C. albicans shows similar growth utilizing fatty acids at both 30°C and 37°C, while Fox3p contributes more significantly to fatty acid utilization at elevated temperatures . These temperature-dependent activity profiles reflect adaptations to host environments and provide insights into the functional specialization of thiolase isoforms.

How can recombinant fadA be used to study antimicrobial resistance mechanisms?

Recombinant fadA provides a valuable tool for investigating antimicrobial resistance mechanisms in several ways:

• Metabolic adaptation studies: Changes in fatty acid metabolism, mediated by thiolases like fadA, can contribute to bacterial adaptation under antibiotic pressure. Recombinant fadA enables controlled studies of these metabolic shifts.

• Resistance gene association: E. fergusonii strains frequently exhibit multidrug resistance, with studies detecting antibiotic-resistant strains in various environments. For example, E. fergusonii isolates from dairy cattle demonstrated resistance to multiple antibiotics including ampicillin, cefoperazone, and trimethoprim-sulfamethoxazole .

• Evolutionary analysis: Recombinant fadA can be used in phylogenetic studies to understand the evolutionary relationship between E. fergusonii and related species, providing context for the emergence of resistant strains.

• Biofilm formation studies: FadA proteins can form amyloid-like structures that may contribute to biofilm development and antibiotic tolerance. Research on pre-FadA demonstrates its role in amyloid-like FadA secretion, which could influence bacterial community formation and resistance .

The emergence of multiple-resistant strains of E. fergusonii highlights the importance of accurate molecular identification methods for tracking these potentially pathogenic bacteria in clinical and environmental settings .

What are the implications of thiolase diversity for metabolic engineering in yeast systems?

The diversity of thiolases in yeast systems offers numerous opportunities for metabolic engineering applications:

Understanding the functional differences between thiolase isoforms enables targeted engineering approaches. For instance, the temperature-dependent contribution of Fox3p to fatty acid utilization suggests applications in processes requiring operation at elevated temperatures . Similarly, the distinct evolutionary origin of Pot13p may provide access to novel substrate specificities not present in conventional yeast thiolases .

What are common challenges in purifying active recombinant E. fergusonii fadA?

Researchers frequently encounter several challenges when purifying active recombinant E. fergusonii fadA:

• Protein solubility issues: Thiolases often form inclusion bodies when overexpressed. Addressing this requires optimization of:

  • Induction temperature (typically lowered to 16-18°C)

  • IPTG concentration (reduced to 0.1-0.5 mM)

  • Co-expression with chaperones

• Loss of activity during purification: Activity loss may result from:

  • Oxidation of catalytic cysteine residues

  • Dissociation of oligomeric structures

  • Removal of essential cofactors

• Structural integrity disruption: Mutations in key residues can dramatically affect protein production and structure. For example, studies on related FadA proteins show that specific mutations (L14A, L76A) completely prevent pre-FadA production and subsequent filamentous structure formation .

• Contamination with host thiolases: When expressing in E. coli, contamination with host thiolases can complicate activity assays. Using thiolase-deficient expression hosts or implementing highly specific purification strategies can address this issue.

How can researchers validate the identity and purity of recombinant fadA preparations?

Comprehensive validation of recombinant fadA identity and purity requires multiple complementary approaches:

Research on E. fergusonii demonstrates that molecular identification methods are essential for reliable differentiation from genetically related species like E. coli . PCR detection methods specifically designed to target characteristic genes provide clear differentiation and are adaptable for safe and rapid use in laboratory settings .

What emerging technologies might advance our understanding of thiolase function across species?

Several cutting-edge technologies offer promising avenues for advancing thiolase research:

• Cryo-electron microscopy: Enabling high-resolution structural analysis of thiolase complexes with substrates and interaction partners without crystallization.

• Single-molecule enzymology: Providing insights into the dynamic behavior of individual thiolase molecules during catalysis, revealing heterogeneity in enzyme populations.

• Systems biology approaches: Integrating thiolase function into whole-cell metabolic models to understand flux control and regulatory networks.

• Comparative genomics and phylogenetics: Expanding our understanding of thiolase evolution through comprehensive analysis of diverse species, as demonstrated in studies comparing C. albicans thiolases with those from other fungi .

• CRISPR-Cas9 genome editing: Enabling precise engineering of thiolase genes in both bacterial and yeast systems to investigate structure-function relationships.

These technologies will help address fundamental questions about thiolase function, such as the evolutionary significance of multiple thiolase isoforms in yeast compared to the single thiolase found in related Saccharomycetales .

How might thiolase research contribute to understanding microbial pathogenesis?

Thiolase research offers several promising pathways for advancing our understanding of microbial pathogenesis:

• Metabolic adaptation during infection: Thiolases play crucial roles in fatty acid metabolism, which may be reprogrammed during host invasion. Understanding these adaptations could reveal new therapeutic targets.

• Biofilm formation: Research demonstrates that FadA proteins can form amyloid-like structures that may contribute to biofilm development . These structures could enhance pathogen persistence and antibiotic tolerance.

• Host-pathogen interactions: Some thiolases may have moonlighting functions beyond their metabolic roles. For instance, FadA can enhance pathogenicity through mechanisms related to its secretion and interaction with host tissues .

• Virulence regulation: Metabolic shifts involving thiolases may trigger virulence factor expression. Studies in C. albicans have investigated the role of thiolases in virulence, though interestingly, deletion of individual thiolases did not significantly attenuate virulence in an embryonated chicken egg infection model .

Continued research on thiolases across microbial species will provide valuable insights into metabolic adaptations during infection and potentially identify novel targets for antimicrobial intervention.