Recombinant Chromobacterium violaceum Fumarate hydratase class II (fumC)

What is Chromobacterium violaceum and why study its fumC enzyme?

Chromobacterium violaceum is a gram-negative bacterium found in tropical and subtropical regions that is generally considered nonpathogenic but can cause severe infections in immunocompromised individuals with high mortality rates . The organism has been documented in approximately 33 cases worldwide since the first human infection was recorded in 1927 . Studying its metabolic enzymes, including fumC, provides insights into bacterial adaptation, pathogenicity mechanisms, and potential biotechnological applications. The class II fumarase (fumC) is particularly interesting because, unlike class I fumarases that participate in DNA damage repair in some organisms, class II fumarases have distinctive catalytic properties and structural characteristics that make them valuable subjects for comparative enzymology.

How do Class I and Class II fumarases differ functionally?

Class I and Class II fumarases represent two evolutionarily distinct enzyme families that catalyze the same reaction but differ significantly in structure and properties:

In organisms like E. coli that possess both classes, Class I fumarases have been demonstrated to participate in DNA damage repair through mechanisms involving alpha-ketoglutarate (α-KG) signaling, which affects DNA damage repair enzymes like AlkB . Though not specifically documented for C. violaceum, similar mechanistic relationships might exist and warrant investigation.

What expression systems are recommended for recombinant C. violaceum fumC?

For recombinant expression of C. violaceum fumC, several expression systems have proven effective, with E. coli being the most commonly utilized host. When designing expression experiments, researchers should consider:

E. coli Expression Systems:

• pET System: Offers high expression levels under T7 promoter control, suitable for large-scale protein production

• pBAD System: Provides more controlled expression through arabinose induction, reducing potential toxicity issues

• pQE System: Facilitates the addition of 6xHis tags for simplified purification protocols

Based on methods used with similar bacterial fumarases, E. coli strain BL21(DE3) or derivatives are generally preferred for expression, with induction typically performed at reduced temperatures (16-25°C) to enhance proper folding and solubility of the recombinant enzyme.

What are typical yields and purification approaches for recombinant fumC?

Typical yields for recombinant C. violaceum fumC vary depending on expression conditions, but researchers can expect 15-25 mg of purified protein per liter of bacterial culture under optimized conditions. A standard purification protocol includes:

• Cell lysis via sonication or pressure disruption in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10 mM imidazole

• Initial purification via Ni-NTA affinity chromatography for His-tagged constructs

• Secondary purification using ion-exchange chromatography (typically Q-Sepharose)

• Final polishing step with size-exclusion chromatography

This approach typically yields >95% pure protein suitable for enzymatic and structural studies.

How can contradictions in experimental data regarding fumC activity be reconciled?

Contradictions in experimental data are a common challenge when studying enzymes like fumC. These discrepancies might arise from several sources:

• Context-specific effects: Research has shown that seemingly contradictory information in scientific knowledge graphs often stems from omission of specific contexts, such as experimental conditions or tissue-specific interactions .

• Methodological differences: Different assay conditions (pH, temperature, buffer composition) can significantly affect enzyme activity measurements.

• Post-translational modifications: Enzyme preparations may contain varied levels of post-translational modifications that affect activity.

• Enzyme purity: Contaminating proteins or enzyme degradation can influence activity measurements.

To reconcile contradictory data, researchers should:

• Implement robust experimental designs with appropriate controls

• Document all experimental conditions meticulously

• Consider using multiple complementary techniques to measure enzyme activity

• Perform statistical analyses to assess data reliability

• Design experiments that specifically address potential contextual factors

The presence of contradictory information has been estimated at about 2.6% in PubMed-scale knowledge graphs, with most apparent contradictions stemming from differences in experimental conditions rather than true scientific disagreements .

What role might C. violaceum fumC play in bacterial pathogenicity?

While the direct role of fumC in C. violaceum pathogenicity remains under investigation, several hypotheses can be formulated based on current understanding:

• Metabolic adaptation: As a TCA cycle enzyme, fumC may contribute to metabolic flexibility during infection, allowing adaptation to host environments.

• Stress response: Class II fumarases tend to be more stable under oxidative stress conditions that may be encountered during host immune responses.

• Potential moonlighting functions: Like other metabolic enzymes, fumC might have secondary functions beyond its catalytic role in the TCA cycle.

C. violaceum infections, while rare, have high mortality rates and occur primarily in immunocompromised individuals . The bacterium's adaptation to host environments may involve metabolic reprogramming that includes altered regulation of TCA cycle enzymes like fumC.

What techniques are recommended for studying potential regulatory mechanisms of fumC expression?

To investigate fumC expression regulation in C. violaceum, researchers can employ several complementary approaches:

• Transcriptional regulation analysis:

  • qRT-PCR or droplet digital PCR (ddPCR) to quantify transcript levels under various conditions

  • Reporter gene assays using the fumC promoter region fused to reporters like lacZ or GFP

• Quorum sensing effects:

  • C. violaceum and related species use quorum sensing systems (like CviI-R in C. substugae) to regulate gene expression in a cell density-dependent manner

  • Evaluation of fumC expression in the presence of homoserine lactones or in quorum sensing mutants

• Stress response regulation:

  • Analysis of fumC expression under various stress conditions (oxidative stress, nutrient limitation)

  • Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the fumC promoter

For transcriptional analysis via ddPCR, researchers should follow protocols similar to those described for C. substugae, using ~1 ng/μL of cDNA template with appropriate primers and reference genes like GAPDH .

How can structural and functional relationships of C. violaceum fumC be systematically investigated?

To elucidate structure-function relationships of C. violaceum fumC, researchers should consider a multifaceted approach:

• Structural determination:

  • X-ray crystallography of purified recombinant enzyme

  • Cryo-electron microscopy for visualization of large assemblies

  • Homology modeling based on related fumarases if experimental structures are unavailable

• Functional characterization:

  • Site-directed mutagenesis of predicted catalytic and regulatory residues

  • Enzyme kinetics studies using spectrophotometric assays

  • Thermal shift assays to assess structural stability of wildtype and mutant proteins

• Protein-protein interaction studies:

  • Pull-down assays to identify interaction partners

  • Surface plasmon resonance to quantify binding affinities

  • Crosslinking mass spectrometry to map interaction interfaces

When designing site-directed mutagenesis experiments, target residues should include those conserved among class II fumarases as well as those unique to C. violaceum fumC.

What experimental designs best assess potential roles of fumC in DNA damage response?

While class I fumarases have been implicated in DNA damage responses in E. coli , the potential role of class II fumarases like C. violaceum fumC remains largely unexplored. To investigate this possibility:

• DNA damage sensitivity assays:

  • Create fumC knockout or knockdown strains

  • Assess sensitivity to DNA-damaging agents (UV, chemical mutagens)

  • Complement with wild-type or mutant fumC to confirm phenotypes

• Metabolite profiling:

  • Measure levels of TCA cycle intermediates during DNA damage

  • Investigate specific metabolites like α-ketoglutarate that might link metabolism to DNA repair

  • Analyze how fumC activity correlates with DNA repair efficiency

• Protein localization studies:

  • Use fluorescent protein fusions to track fumC localization during normal growth and DNA damage

  • Perform subcellular fractionation and immunoblotting

• Interaction studies with DNA repair enzymes:

  • Investigate potential interactions between fumC and DNA repair proteins

  • Assess the impact of fumC metabolites (fumarate, malate) on DNA repair enzyme activities

In E. coli, class I fumarases participate in DNA damage repair through mechanisms involving α-KG and AlkB . Researchers should design experiments to determine if C. violaceum fumC influences similar pathways or acts through alternative mechanisms.

What are optimal enzyme activity assay conditions for recombinant C. violaceum fumC?

The standard fumarase activity assay measures the conversion of fumarate to L-malate (or vice versa) spectrophotometrically. For C. violaceum fumC:

Forward Reaction (Fumarate → L-malate):

• Buffer: 50 mM potassium phosphate, pH 7.4-7.6

• Temperature: 25-30°C (optimal for most measurements)

• Substrate: 1-10 mM fumarate

• Detection: Monitor decrease in absorbance at 240 nm (ε = 2.53 mM⁻¹cm⁻¹)

Reverse Reaction (L-malate → Fumarate):

• Buffer: 50 mM potassium phosphate, pH 7.4-7.6

• Temperature: 25-30°C

• Substrate: 10-50 mM L-malate

• Detection: Monitor increase in absorbance at 240 nm

For both assays, enzyme concentration should be adjusted to ensure linear reaction rates, typically in the range of 0.5-5 μg/ml of purified enzyme.

How can researchers address solubility and stability challenges with recombinant fumC?

Recombinant C. violaceum fumC, like many bacterial enzymes, may present solubility and stability challenges during expression and purification. To address these issues:

Improving Solubility:

• Lower induction temperature (16-18°C)

• Reduce inducer concentration

• Co-express with molecular chaperones (GroEL/GroES)

• Use solubility-enhancing fusion tags (SUMO, MBP, TRX)

• Optimize buffer conditions (add glycerol, low concentrations of detergents)

Enhancing Stability:

• Include stabilizing agents in storage buffer (10-20% glycerol)

• Determine optimal pH range (typically pH 7.0-8.0)

• Add reducing agents if cysteine residues are present (DTT or β-mercaptoethanol)

• Store at -80°C in small aliquots to avoid freeze-thaw cycles

What approaches can resolve contradictions between in vitro and in vivo fumC activity data?

Discrepancies between in vitro enzyme measurements and in vivo activity are common challenges in enzyme research. To address these contradictions:

• Develop cell-based assay systems:

  • Create reporter systems linked to fumC activity

  • Use metabolomics to track fumarate/malate ratios in vivo

• Consider physiological context:

  • Measure enzyme activity under conditions that mimic the bacterial cytoplasm

  • Include potential physiological regulators in in vitro assays

  • Account for substrate availability and transport in vivo

• Examine post-translational modifications:

  • Compare enzyme purified from native source versus recombinant systems

  • Identify potential modifications using mass spectrometry

• Investigate protein-protein interactions:

  • Identify potential interaction partners that might modulate activity

  • Reconstitute multiprotein complexes in vitro

How might high-throughput approaches advance understanding of C. violaceum fumC?

High-throughput methodologies offer significant potential for advancing understanding of C. violaceum fumC:

These approaches can generate large datasets that require careful analysis to resolve apparent contradictions, a common challenge in scientific knowledge graphs derived from biomedical literature .

What is known about potential interactions between fumC and quorum sensing in Chromobacterium species?

While direct evidence for interactions between fumC and quorum sensing in C. violaceum is limited, related species provide interesting insights:

C. substugae utilizes a LuxI-R-type quorum-sensing system (CviI-R) that regulates gene expression in a cell density-dependent manner . CviI synthesizes N-hexanoyl-homoserine lactone (C6-HSL), and CviR is a C6-HSL-responsive transcription regulator that activates numerous genes .

Potential experimental approaches to investigate fumC-quorum sensing interactions include:

• Analysis of fumC expression in quorum sensing mutants

• Examination of fumC promoter regions for potential binding sites for quorum sensing regulators

• Metabolic profiling to determine if TCA cycle intermediates influence quorum sensing pathways

For transcriptional studies, methods similar to those used for the cdeA promoter in C. substugae might be applied to fumC, using β-galactosidase reporter assays or droplet digital PCR for transcript quantification .