Recombinant Serratia proteamaculans 6-phosphofructokinase (pfkA)

What is Serratia proteamaculans 6-phosphofructokinase (pfkA) and what is its metabolic significance?

Serratia proteamaculans 6-phosphofructokinase (pfkA) is a key enzyme in the glycolytic pathway that catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate using ATP as a phosphoryl donor. This reaction represents the first committing step of glycolysis, making it a critical control point in carbohydrate metabolism . In bacterial species like S. proteamaculans, pfkA plays an essential role in energy production, particularly when the organism relies heavily on glycolysis for ATP generation.

The enzyme's activity significantly impacts the metabolic flux through glycolysis, influencing downstream pathways including the TCA cycle and pentose phosphate pathway. Like other bacterial phosphofructokinases, S. proteamaculans pfkA is likely subject to allosteric regulation, responding to cellular energy levels and metabolic intermediates to maintain appropriate glycolytic flux.

How can researchers reliably measure S. proteamaculans pfkA activity in laboratory settings?

Several established methods can be used to measure S. proteamaculans pfkA activity:

• Coupled spectrophotometric assays: The most common approach links the production of fructose 1,6-bisphosphate to the oxidation of NADH, which can be monitored at 340 nm. This typically involves coupling the PFK reaction with aldolase, triosephosphate isomerase, and glycerol-3-phosphate dehydrogenase.

• Commercial activity assay kits: Colorimetric methods, such as those referenced in the search results, can detect phosphofructokinase activity with high sensitivity (less than 1 mU) . These kits offer standardized protocols that reduce technical variability between experiments.

• Standard reaction conditions: Typical reaction mixtures include a buffer system (often HEPES or Tris at pH 7.5-8.0), ATP (1-2 mM), fructose 6-phosphate (1-2 mM), and MgCl₂ (5-10 mM) as a cofactor. Temperature optimization is crucial, with bacterial PFKs typically showing optimal activity between 25-37°C.

When measuring activity in crude extracts, it's important to account for potential interference from other enzymes by including appropriate controls.

What expression systems are most effective for producing recombinant S. proteamaculans pfkA?

For recombinant production of S. proteamaculans pfkA, several expression systems can be employed:

• E. coli expression systems: These typically offer the highest yields and simplest purification workflows. Specialized pfkA-deficient E. coli strains like RL257 (which has both pfkA and pfkB deleted) are particularly valuable as they eliminate background phosphofructokinase activity that might interfere with subsequent enzyme characterization .

• Inducible expression vectors: Vectors containing inducible promoters such as T7 or tac, coupled with the lacI(q) system present in many laboratory strains, allow for controlled expression of the enzyme . This is especially important for potentially toxic proteins.

• Affinity tags: For structural studies requiring high protein purity, fusion tags such as His₆ or GST can facilitate purification via affinity chromatography.

• Expression conditions: Cold-shock expression protocols are sometimes beneficial for improving the solubility of recombinant PFK enzymes, as they slow protein synthesis and may enhance proper folding.

When native regulatory properties are being studied, care must be taken to remove any potential allosteric effectors during the purification process.

What are the optimal storage conditions for preserving recombinant S. proteamaculans pfkA activity?

To maintain optimal activity of recombinant S. proteamaculans pfkA, proper storage conditions are critical:

• Temperature: Purified enzyme preparations should be stored at -80°C for long-term preservation, preferably in small aliquots to avoid repeated freeze-thaw cycles that can lead to significant activity loss.

• Buffer composition: A stabilizing buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100 mM KCl, 5 mM MgCl₂, and 1-5 mM DTT or 2-mercaptoethanol helps maintain enzyme structure and function.

• Cryoprotectants: The addition of glycerol (20-50%) serves as a cryoprotectant and further stabilizes the enzyme during freezing.

• Short-term storage: For periods of 1-2 weeks, the enzyme can be kept at 4°C with preservatives such as 0.02% sodium azide to prevent microbial contamination.

• Activity verification: Before use, activity retention should be verified using standardized assay conditions to establish the effective shelf-life under the chosen storage parameters.

How do the kinetic parameters of S. proteamaculans pfkA compare with those of other bacterial phosphofructokinases?

Bacterial phosphofructokinases exhibit considerable variation in their kinetic parameters, reflecting adaptations to different environmental niches and metabolic demands:

• Michaelis-Menten kinetics: Most bacterial PFKs, including those from Serratia species, display Michaelis-Menten kinetics with Km values for fructose 6-phosphate typically ranging from 0.05-0.3 mM and for ATP from 0.1-0.5 mM.

• Catalytic efficiency: The catalytic efficiency (kcat/Km) of bacterial PFKs generally exceeds that of eukaryotic counterparts, reflecting the central role of glycolysis in bacterial energy metabolism.

• Allosteric regulation: Unlike eukaryotic PFKs that are inhibited by ATP, bacterial enzymes including those from Serratia species are typically not inhibited by high ATP concentrations, allowing continued glycolytic flux even under energy-rich conditions.

• Activators and inhibitors: S. proteamaculans pfkA likely shows allosteric activation by ADP and GDP, similar to other Enterobacteriaceae PFKs. Phosphoenolpyruvate (PEP) may serve as a feedback inhibitor, though the sensitivity to this inhibition varies considerably among bacterial species.

Comparative enzyme kinetics studies would be valuable to determine the specific parameters for S. proteamaculans pfkA and how they relate to the organism's ecological niche.

What is the relationship between S. proteamaculans pfkA expression and bacterial virulence?

The relationship between S. proteamaculans pfkA expression and bacterial virulence appears complex and multifaceted:

• Growth phase correlation: In Serratia species, invasive activity typically appears at the stationary growth phase, corresponding to maximal bacterial population density . This timing coincides with metabolic adaptations, including shifts in glycolytic enzyme expression.

• Metabolic impact on virulence factors: Phosphofructokinase activity can significantly affect the balance between glycolysis and the pentose phosphate pathway, thereby influencing NADPH production that fuels important virulence mechanisms like oxidative stress resistance .

• Quorum sensing connections: In S. proteamaculans, the quorum sensing (QS) system regulates invasive activity through mechanisms that may involve central metabolic pathways . The inactivation of sprI in S. proteamaculans leads to increased invasive activity , suggesting potential metabolic rewiring that could involve altered pfkA expression or activity.

• Moonlighting functions: Glycolytic enzymes including PFK can potentially moonlight as adhesins or immunomodulatory proteins when expressed on the bacterial surface.

Understanding the precise relationship between pfkA and virulence would require analyzing gene expression patterns during infection, constructing pfkA knockout mutants, and performing virulence assays under different metabolic conditions.

How does the quorum sensing system in S. proteamaculans influence pfkA expression and activity?

The quorum sensing (QS) system in S. proteamaculans likely influences pfkA expression and activity as part of a broader metabolic regulation network:

• QS system components: S. proteamaculans employs a LuxI/LuxR type QS system consisting of the regulatory protein SprR and the AHL synthase SprI .

• Metabolic reprogramming: The inactivation of the sprI gene results in significant changes to the bacterium's invasive properties , suggesting extensive rewiring of metabolic and virulence pathways that may include glycolytic enzymes like pfkA.

• Population density effects: As bacteria transition to stationary phase and accumulate autoinducers, metabolic priorities shift from rapid growth to persistence and virulence. This transition typically involves changes in glycolytic flux, where pfkA plays a pivotal regulatory role.

• Regulatory mechanisms: The QS system may regulate pfkA at multiple levels: transcriptionally through direct or indirect binding of SprR to the pfkA promoter; post-transcriptionally through regulatory RNAs; or post-translationally by influencing protein modification or stability.

• Experimental approaches: Comparing pfkA expression and activity in wild-type versus sprI mutant strains , chromatin immunoprecipitation to detect SprR binding to the pfkA promoter, and metabolic flux analysis could help elucidate these regulatory connections.

What methodological approaches can be used to investigate the role of pfkA in S. proteamaculans adaptation to different carbon sources?

Investigating the role of pfkA in S. proteamaculans adaptation to different carbon sources requires a comprehensive methodological toolkit:

• Genetic manipulation:

  • Gene deletion or CRISPR-Cas9 mediated knockout of pfkA to establish its essentiality across various carbon substrates

  • Complementation studies using controlled expression vectors

  • Site-directed mutagenesis for structure-function analyses

• Expression analysis:

  • Transcriptional analysis via RT-qPCR or RNA-seq to reveal how pfkA expression responds to different carbon sources

  • Proteomics to identify post-translational modifications affecting enzyme activity

• Metabolic studies:

  • Metabolic flux analysis using ¹³C-labeled substrates to quantify carbon flow through glycolysis versus alternative pathways

  • Intracellular metabolite concentration measurements via LC-MS/MS to identify potential feedback mechanisms

  • Growth kinetics in defined media with various carbon sources

• Biochemical characterization:

  • Enzyme kinetics with purified recombinant pfkA to determine substrate preferences and regulatory properties

  • High-throughput activity assays for screening conditions

  • Isothermal titration calorimetry to characterize interactions with allosteric effectors

• Comparative approaches:

  • Genomic comparisons across Serratia species occupying different ecological niches

  • Heterologous expression of pfkA variants in model systems like the RL257 E. coli strain

What are the challenges in crystallizing recombinant S. proteamaculans pfkA for X-ray diffraction studies?

Crystallizing recombinant S. proteamaculans pfkA for X-ray diffraction studies presents several significant challenges:

• Protein quality:

  • Obtaining highly pure, homogeneous, and conformationally stable enzyme preparations

  • Bacterial PFKs typically form homotetramers with considerable conformational flexibility

  • Expression systems may require optimization to minimize aggregation

• Conformational heterogeneity:

  • The allosteric nature of PFK means it can adopt multiple conformational states

  • Co-crystallization with substrate analogs or allosteric effectors may be necessary to lock the enzyme in a defined state

• Expression considerations:

  • E. coli strains lacking endogenous PFK activity, such as RL257 , can be valuable to prevent contamination with host enzyme

  • Tag removal using specific proteases might be necessary if fusion tags interfere with crystallization

• Crystallization conditions:

  • Extensive screening of buffer conditions, precipitants, and additives

  • Surface entropy reduction through site-directed mutagenesis may enhance crystal packing

  • Temperature, pH, and ionic strength optimization

  • Consideration of the natural environment of S. proteamaculans enzymes

• Data collection and analysis:

  • Large unit cells due to the tetrameric nature of the enzyme may require synchrotron radiation

  • Phase determination challenges if molecular replacement with homologous structures is insufficient

How can researchers design experiments to distinguish between pfkA and pfkB activity in S. proteamaculans?

Distinguishing between pfkA and pfkB activities in S. proteamaculans requires careful experimental design:

• Genetic approaches:

  • Construction of single and double knockout strains (ΔpfkA, ΔpfkB, and ΔpfkA/ΔpfkB)

  • Complementation with individual genes to confirm phenotypes

  • Similar approaches have been used successfully in E. coli studies

• Biochemical differentiation:

  • Differential sensitivity to allosteric regulators (pfkA is typically more responsive to activators like ADP)

  • Substrate affinity differences (pfkA generally has higher affinity for fructose 6-phosphate)

  • Different pH optima and divalent cation requirements

• Expression analysis:

  • Gene-specific probes for Northern blotting or RT-qPCR

  • Proteomic approaches using peptide mass fingerprinting

  • Western blotting with isoform-specific antibodies if available

• Heterologous expression:

  • Expression in a PFK-deficient background like the RL257 E. coli strain

  • Purification and characterization of individual enzymes

• Activity assays:

  • Commercial phosphofructokinase activity assay kits can be adapted to maximize specificity

  • Controlled reaction conditions can sometimes favor one isoform over the other

This systematic approach would allow researchers to accurately characterize the relative contributions of pfkA and pfkB to S. proteamaculans metabolism.

What are the best protocols for analyzing the regulatory effects of metabolites on S. proteamaculans pfkA activity?

To thoroughly analyze metabolite regulation of S. proteamaculans pfkA activity:

• Enzyme preparation:

  • Highly purified recombinant enzyme using expression systems like those described for E. coli

  • Removal of bound metabolites during purification

  • Verification of homogeneity and activity

• Screening approach:

  • Initial broad screening of potential regulators (ATP, ADP, AMP, PEP, citrate, fructose 2,6-bisphosphate)

  • Concentration-dependent effects using colorimetric activity assays

  • Construction of activation/inhibition curves

• Kinetic characterization:

  • Determination of mechanism (competitive, non-competitive, uncompetitive)

  • Calculation of Ki or Ka values

  • Synergistic or antagonistic effects of multiple regulators

• Structural confirmation:

  • Isothermal titration calorimetry to confirm direct binding

  • Hydrogen-deuterium exchange mass spectrometry to identify conformational changes

  • Site-directed mutagenesis of putative allosteric sites

• Physiological relevance:

  • Correlation of in vitro findings with metabolomic data

  • Effects of environmental conditions on metabolite levels and enzyme activity

  • Comparison with regulatory patterns in related Serratia species

This comprehensive approach would provide a detailed understanding of the complex allosteric regulation of S. proteamaculans pfkA.

How should researchers interpret contradictory results when studying S. proteamaculans pfkA in different experimental systems?

When faced with contradictory results in S. proteamaculans pfkA studies:

• Experimental context evaluation:

  • Different expression systems may yield enzymes with varying post-translational modifications

  • Growth conditions affect cellular physiology and potentially enzyme behavior

  • Presence of contaminating proteins or metabolites in preparations

• Methodological considerations:

  • Assay sensitivity and specificity differences

  • Buffer components that might influence activity

  • Temperature and pH variations between studies

• Genetic factors:

  • Strain-specific variations in S. proteamaculans

  • Potential compensatory mechanisms in knockout studies

  • Polar effects in genetic constructs

• Regulatory complexity:

  • Different metabolic states may trigger different regulatory mechanisms

  • Quorum sensing effects on metabolism may vary with population density

  • Invasive vs. non-invasive growth contexts may show different pfkA regulation

• Resolution approaches:

  • Direct side-by-side comparison using standardized methods

  • Collaboration between laboratories to identify variables

  • Meta-analysis of published data with attention to methodological details

  • Development of consensus protocols for studying S. proteamaculans pfkA

Understanding these factors can help researchers reconcile seemingly contradictory results and develop a more nuanced understanding of this enzyme's behavior.

What bioinformatic approaches are most effective for analyzing S. proteamaculans pfkA sequence and structure?

For comprehensive bioinformatic analysis of S. proteamaculans pfkA:

• Sequence analysis:

  • Multiple sequence alignment with pfkA from related species

  • Identification of conserved catalytic and regulatory domains

  • Phylogenetic analysis to understand evolutionary relationships

  • Codon usage analysis to optimize heterologous expression

• Structural prediction and analysis:

  • Homology modeling based on crystallized bacterial PFKs

  • Molecular dynamics simulations to predict conformational changes

  • Identification of potential allosteric sites

  • Protein-protein interaction interface prediction for quaternary structure

• Functional prediction:

  • Catalytic site conservation analysis

  • Prediction of post-translational modification sites

  • Identification of potential regulatory elements in the gene promoter

  • Metabolic network modeling to predict systemic effects of pfkA alterations

• Tools and resources:

  • Specialized structure prediction tools like AlphaFold

  • Molecular visualization software for structural analysis

  • Metabolic pathway databases for context

  • Bacterial genome repositories for comparative genomics

• Integration with experimental data:

  • Incorporation of mutagenesis results to refine models

  • Using activity data to validate structural predictions

  • Correlating expression patterns with predicted regulatory elements

These approaches provide a foundation for understanding S. proteamaculans pfkA at the molecular level and guide experimental design.

What are the most promising applications of S. proteamaculans pfkA in metabolic engineering?

S. proteamaculans pfkA offers several promising applications in metabolic engineering:

• Enhanced glycolytic flux:

  • Expression of S. proteamaculans pfkA in industrial microorganisms to overcome rate-limiting steps in glycolysis

  • Fine-tuning of expression levels to optimize carbon utilization

  • Creation of pfkA variants with altered regulatory properties

• Pathway optimization:

  • Balancing glycolytic flux with pentose phosphate pathway activity

  • Redirecting carbon flow for production of high-value metabolites

  • Reducing overflow metabolism in industrial fermentations

• Stress resistance:

  • Engineering improved metabolic responses to environmental stresses

  • Enhancing NADPH production via complementary pathway engineering

  • Improving growth on alternative carbon sources

• Synthetic biology applications:

  • Incorporation into minimal synthetic cells

  • Development of metabolic biosensors based on pfkA regulation

  • Creation of orthogonal glycolytic pathways

• Expression system improvements:

  • Development of pfkA-based selection systems for molecular biology

  • Creation of specialized host strains like the RL257 E. coli model

  • Tunable expression systems using pfkA regulatory elements

These applications could significantly impact industrial biotechnology, biofuel production, and synthetic biology research.

How might understanding S. proteamaculans pfkA contribute to novel antimicrobial development?

Understanding S. proteamaculans pfkA could contribute to antimicrobial development in several ways:

• Target validation:

  • Determining the essentiality of pfkA in S. proteamaculans under different conditions

  • Evaluating pfkA as a potential therapeutic target

  • Identifying structural or regulatory features unique to bacterial PFKs

• Inhibitor development:

  • Structure-based design of pfkA inhibitors

  • Allosteric inhibitors targeting regulatory sites

  • Species-selective inhibitors based on structural differences

• Virulence modulation:

  • Targeting the metabolic basis of virulence without killing bacteria directly

  • Inhibiting the relationship between metabolism and invasiveness

  • Disrupting quorum sensing-regulated metabolic shifts

• Resistance considerations:

  • Understanding potential resistance mechanisms

  • Identification of compensatory pathways

  • Combination approaches targeting multiple metabolic enzymes

• Alternative approaches:

  • Immunomodulatory strategies targeting moonlighting functions of pfkA

  • Developing compounds that affect S. proteamaculans pfkA regulation rather than activity

  • Creating metabolic sensitizers that make bacteria more susceptible to existing antibiotics

Given the increasing problem of antibiotic resistance, novel targets like specialized metabolic enzymes may offer valuable new therapeutic approaches.