Recombinant Aliivibrio salmonicida 6-phosphofructokinase (pfkA)

What is the significance of pfkA in Aliivibrio salmonicida metabolism?

PfkA constitutes a critical enzyme in the glycolytic pathway of A. salmonicida, catalyzing an irreversible and rate-limiting step in glycolysis. This enzyme is particularly significant in A. salmonicida as it plays a central role in carbohydrate utilization during various growth phases and environmental conditions. Research suggests that pfkA activity is integrated with the organism's adaptation mechanisms for survival in different environments, including during host infection. The enzyme's homology domains have been identified in regulatory proteins like RapZ, suggesting evolutionary repurposing of enzyme components from central metabolism for regulatory functions . This evolutionary connection implies pfkA's ancient and essential role in A. salmonicida's metabolic network.

How does pfkA expression relate to pathogenicity in A. salmonicida?

Aliivibrio salmonicida is the causative agent of cold-water vibriosis affecting farmed fish species, a disease that today is effectively controlled by vaccination . Although direct evidence linking pfkA expression to virulence is limited, several aspects of metabolism regulated by pfkA may influence pathogenicity. As central carbon metabolism is often connected to virulence factor production in pathogens, pfkA likely plays an indirect role in pathogenicity. Carbon flux through glycolysis, regulated by pfkA, provides essential energy and metabolic intermediates for various cellular processes, including those related to virulence. Research on related Vibrio species suggests that metabolic adaptation, particularly carbohydrate utilization pathways where pfkA functions, can significantly impact virulence gene expression during host colonization.

What distinguishes A. salmonicida pfkA from other bacterial phosphofructokinases?

While sharing the basic catalytic function with other bacterial phosphofructokinases, A. salmonicida pfkA exhibits distinct characteristics reflecting adaptation to the organism's ecological niche as a cold-water fish pathogen. The enzyme likely possesses adaptations for function at lower temperatures corresponding to the organism's environment. Structural analysis suggests that A. salmonicida pfkA may have unique regulatory properties compared to homologs from mesophilic bacteria. Interestingly, regulatory elements of pfkA appear to have been evolutionary repurposed, as evidenced by the homology between the C-terminus of regulatory protein RapZ and a subdomain of 6-phosphofructokinase .

What are optimal conditions for expressing recombinant A. salmonicida pfkA?

For optimal expression of recombinant A. salmonicida pfkA, consider the following parameters:

Expression System: E. coli BL21(DE3) typically yields good expression levels for bacterial enzymes like pfkA. Alternative hosts include E. coli Arctic Express for cold-adapted protein expression, which may be particularly relevant for this cold-water pathogen enzyme.

Temperature Considerations: Given A. salmonicida's nature as a cold-water pathogen, expression at lower temperatures (15-20°C) after induction often produces more soluble and active enzyme compared to standard expression protocols at 37°C. This approach reduces inclusion body formation and preserves enzymatic activity.

Induction Parameters: Using lower IPTG concentrations (0.1-0.5 mM) and longer induction times (12-16 hours) at reduced temperatures generally improves yield of active protein. The metabolic characteristics of A. salmonicida as a cold-water organism suggest its proteins may fold more efficiently at lower temperatures .

Media Composition: Enriched media such as Terrific Broth supplemented with glucose may enhance yields by providing abundant precursors for protein synthesis while supporting energy metabolism through glycolysis.

What purification strategies yield highest activity for recombinant A. salmonicida pfkA?

Effective purification of recombinant A. salmonicida pfkA requires a strategy that preserves enzymatic activity while achieving high purity:

Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag is generally effective for initial capture. Buffer considerations should include:

• Maintaining pH 7.0-8.0

• Including glycerol (10-15%) to stabilize the enzyme

• Adding reducing agents like DTT or β-mercaptoethanol (1-5 mM) to prevent oxidation

• Incorporating substrate analogs or phosphate (1-5 mM) for additional stability

Secondary Purification: Size exclusion chromatography separates active oligomeric forms from aggregates and further removes contaminants. For highest recovery of active enzyme, all purification steps should be performed at 4-10°C, respecting the cold-adapted nature of A. salmonicida proteins.

Activity Preservation: Adding stabilizing agents such as ammonium sulfate at moderate concentrations (50-200 mM) often helps maintain activity during purification and storage. Kinetic analyses show that A. salmonicida enzymes generally display higher catalytic efficiency at lower temperatures compared to mesophilic counterparts, reflecting their adaptation to cold environments.

How can enzyme kinetics of A. salmonicida pfkA be accurately measured?

Accurate kinetic measurements of A. salmonicida pfkA require consideration of its cold-adapted nature and potential regulatory properties:

Temperature Range: Assays should be performed across multiple temperatures (4-30°C) to determine temperature optima and capture the enzyme's psychrophilic characteristics. This is particularly important as A. salmonicida has evolved for functioning in cold marine environments .

Data Analysis: Apply non-linear regression to determine kinetic parameters (Km, Vmax, Hill coefficients). For cold-adapted enzymes like those from A. salmonicida, Arrhenius plots often show deviations indicating different activation energies compared to mesophilic homologs.

How does temperature influence A. salmonicida pfkA activity and stability?

A. salmonicida pfkA, as an enzyme from a cold-water pathogen, exhibits distinct temperature-dependent behaviors:

Activity Profile: The enzyme typically shows higher catalytic activity (kcat) at low temperatures (4-15°C) compared to mesophilic homologs, with a temperature optimum likely in the 15-20°C range. This corresponds to the natural habitat temperature range of A. salmonicida.

Cold Adaptation Features: Structural analysis would likely reveal characteristic features of cold-adapted enzymes, including:

• Reduced number of salt bridges and hydrogen bonds

• Increased surface hydrophobicity

• More flexible active site regions

• Lower activation enthalpy

Thermal Stability: A. salmonicida pfkA demonstrates reduced thermal stability compared to mesophilic counterparts, with significant activity loss typically occurring above 25-30°C. This reduced stability represents an evolutionary trade-off for enhanced catalytic efficiency at lower temperatures.

Temperature-Dependent Regulation: Temperature shifts likely influence pfkA expression and activity as part of A. salmonicida's adaptation to environmental changes. Research on A. salmonicida has shown that temperature influences various regulatory systems, including those controlling biofilm formation , suggesting metabolic enzymes like pfkA may also be subject to temperature-dependent regulation.

What is the relationship between pfkA activity and quorum sensing in A. salmonicida?

The relationship between pfkA activity and quorum sensing in A. salmonicida involves complex regulatory networks:

Metabolic Integration: Quorum sensing (QS) systems in A. salmonicida, particularly the LuxI-LuxR and AinS-AinR systems, regulate numerous physiological processes including biofilm formation and virulence . These systems are likely integrated with central carbon metabolism, where pfkA functions as a key regulatory enzyme.

Regulatory Mechanisms: Research indicates that Spot 42, a small regulatory RNA in A. salmonicida, impacts carbohydrate metabolism and is regulated by cAMP-CRP similar to E. coli . This suggests a potential regulatory connection between quorum sensing, carbon catabolite repression, and glycolytic flux through pfkA.

Expression Dynamics: Transcriptomic analyses have shown that quorum sensing mutants (ΔlitR and ΔrpoQ) in A. salmonicida display differential expression of genes involved in motility, adhesion, and biofilm formation . While direct evidence for pfkA regulation is limited, metabolic enzymes often show expression changes in response to quorum sensing signals, reflecting shifts in energy utilization during different growth phases.

Biofilm Connection: Since biofilm formation requires significant metabolic adaptation and A. salmonicida forms structured biofilms , pfkA activity likely changes during biofilm development to accommodate altered energy demands and carbon flux distributions.

How can metabolic flux analysis be applied to study pfkA function in A. salmonicida?

Metabolic flux analysis provides powerful approaches to understand pfkA's role in A. salmonicida metabolism:

Dynamic FBA Applications: Dynamic FBA (dFBA) approaches allow modeling of temporal metabolic shifts, such as transitions between different carbon sources . This is particularly relevant for understanding how A. salmonicida adapts its metabolism during infection or environmental changes.

Experimental Validation: Combine computational predictions with experimental measurements using:

• Isotope labeling experiments with 13C-glucose to track carbon flux through glycolysis

• Metabolomics to measure changes in glycolytic intermediates

• Enzyme activity assays under various conditions

Subpopulation Considerations: Recent advances in metabolic modeling incorporate population heterogeneity , which may be important when studying A. salmonicida metabolism in biofilms or during host infection, where metabolically distinct subpopulations might emerge.

What structural features contribute to A. salmonicida pfkA function in cold environments?

A. salmonicida pfkA likely possesses several structural adaptations that facilitate function in cold environments:

Active Site Flexibility: Cold-adapted enzymes typically feature more flexible active sites that require lower activation energy, facilitating catalysis at lower temperatures. This flexibility often comes from reduced proline content in loop regions and fewer rigid cross-links near the active site.

Surface Properties: A. salmonicida pfkA would be expected to have:

• Increased surface hydrophobicity

• Reduced number of arginine residues

• Increased glycine content in loop regions

• Modified electrostatic surface potential compared to mesophilic homologs

Oligomeric Stability: While bacterial pfkA typically forms tetramers, the subunit interactions in A. salmonicida pfkA may be modified to balance structural stability with catalytic flexibility at low temperatures. Notably, structural analysis has shown that related proteins like RapZ form unusual quaternary structures comprising domain-swapped dimer-of-dimers arrangements , suggesting complex structural evolution in this protein family.

Ligand Binding: Cold adaptation often modifies ligand binding properties, potentially affecting both substrate affinity and allosteric regulation. The binding pocket identified in RapZ, which has homology to a subdomain of phosphofructokinase, suggests interesting evolutionary relationships between metabolic enzymes and regulatory proteins .

How does site-directed mutagenesis help elucidate A. salmonicida pfkA catalytic mechanism?

Site-directed mutagenesis provides critical insights into A. salmonicida pfkA function:

Catalytic Residues: Mutation of predicted catalytic residues (typically aspartate, glutamate, and arginine residues in the active site) allows verification of their roles in substrate binding and catalysis. Comparison of kinetic parameters between wild-type and mutant enzymes reveals the contribution of specific residues to the catalytic efficiency.

Cold Adaptation Features: Strategic mutations targeting residues unique to A. salmonicida pfkA compared to mesophilic homologs can identify specific adaptations for cold activity. Replacement of flexible glycine residues with more rigid proline, for example, would test hypotheses about local flexibility contributing to cold adaptation.

Allosteric Regulation: Mutations in predicted regulatory binding sites help map allosteric mechanisms specific to A. salmonicida pfkA. This approach has successfully identified regulatory mechanisms in other bacterial phosphofructokinases and could reveal unique features in this cold-adapted enzyme.

Experimental Design: A comprehensive mutagenesis study would include:

• Sequence alignment with homologs to identify conserved and unique residues

• Structural modeling to predict effects of mutations

• Production of mutant variants using standard molecular biology techniques

• Comparative kinetic analysis across temperature ranges

• Stability studies to separate effects on catalysis from effects on protein stability

How can contradictory results in pfkA activity assays be reconciled?

Researchers often encounter contradictory results when measuring pfkA activity, particularly with cold-adapted enzymes. These inconsistencies can be systematically addressed:

Temperature Effects: Cold-adapted enzymes like A. salmonicida pfkA are particularly sensitive to temperature during assays. Apparently contradictory measurements often result from small temperature variations between experiments. Implementing strict temperature control and reporting exact assay temperatures is essential.

Buffer Composition Impact: Even minor differences in buffer composition can significantly affect activity measurements. Key factors include:

• pH (even 0.2 unit differences matter)

• Ionic strength

• Presence of stabilizing agents

• Metal ion concentrations (particularly Mg2+)

Assay Methodology Differences: Different coupling systems or direct assays may yield varying results. When comparing literature values or troubleshooting contradictory findings, carefully consider methodological differences. The minimization of metabolic adjustment (MOMA) approach used in some metabolic studies highlights how different optimization criteria can yield different flux distributions.

Statistical Approach: Apply robust statistical analyses to distinguish significant differences from experimental noise:

• Use sufficient replicates (minimum n=3, preferably n≥5)

• Apply appropriate statistical tests (ANOVA for multiple comparisons)

• Report confidence intervals rather than just p-values

• Consider Bayesian approaches for integrating prior knowledge with new measurements

What approaches help link pfkA activity to broader metabolic networks in A. salmonicida?

Understanding pfkA's role in broader metabolic contexts requires integrative approaches:

Multi-omics Integration: Combine multiple data types to build comprehensive metabolic models:

• Transcriptomics data reveals expression patterns under different conditions

• Proteomics confirms enzyme abundance

• Metabolomics tracks changes in metabolite pools

• Fluxomics measures actual carbon flow

Pathway Analysis: Techniques like Flux Balance Analysis (FBA), parsimonious FBA (pFBA), and minimization of metabolic adjustment (MOMA) help predict how changes in pfkA activity ripple through metabolism . These computational approaches simulate the impact of pfkA modulation on growth rate, byproduct formation, and energy generation.

Network Perturbation: Experimental perturbations through:

• Gene knockdown/knockout studies

• Enzyme inhibition

• Environmental shifts (temperature, pH, carbon source)
  provide essential validation of computational predictions.

Physiological Correlation: Connect enzyme-level observations to organism-level phenotypes by examining how changes in pfkA activity correlate with:

• Growth rate under different conditions

• Biofilm formation capacity

• Virulence in infection models

• Stress response capabilities