Recombinant Escherichia coli O45:K1 6-phosphofructokinase (pfkA)

What is phosphofructokinase (pfkA) and what role does it play in E. coli metabolism?

Phosphofructokinase I (PFK I) is an extensively studied allosteric enzyme encoded by the pfkA gene in Escherichia coli . It catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP), which represents the first committed step in glycolysis. This reaction is a major control point in central carbon metabolism, making PFK I crucial for regulating glycolytic flux. The enzyme functions as a metabolic sensor, responding to the energy status of the cell through various allosteric mechanisms. PFK I exhibits cooperative interactions with its substrate fructose-6-phosphate, is inhibited by phosphoenolpyruvate (PEP), and is activated by ADP . These regulatory properties allow E. coli to adjust glycolytic flux in response to changing energy demands and nutrient availability, making pfkA essential for metabolic adaptation across different environmental conditions.

What are the main differences between PFK I (pfkA) and PFK II (pfkB) in E. coli?

E. coli possesses two distinct phosphofructokinase isozymes with significant differences in their structural and functional properties. PFK I, encoded by pfkA, is the main phosphofructokinase and accounts for approximately 90% of total PFK activity under normal growth conditions . In contrast, PFK II, encoded by pfkB, is normally a minor isozyme that becomes more significant in strains carrying the pfkB1 mutation (a suppressor of pfkA mutants) .

The two isozymes differ significantly in their regulatory properties. PFK I is an allosteric enzyme that shows cooperative interactions with fructose-6-P, is inhibited by phosphoenolpyruvate (PEP), and is activated by ADP . Conversely, PFK II is non-allosteric, showing no cooperative interactions with fructose-6-P and no response to the same allosteric modulators that affect PFK I . Structurally, both enzymes function as tetramers, but PFK II has a slightly larger subunit molecular weight than PFK I (36,000 and 34,000 Da, respectively) .

Another notable difference is that PFK II, unlike PFK I, is somewhat sensitive to inhibition by fructose-1,6-bisphosphate and can use tagatose-6-P as an alternative substrate . No immunological cross-reactivity has been detected between the two isozymes, indicating substantial differences in their protein structures . This molecular diversity provides E. coli with metabolic flexibility, allowing glycolysis to continue even when PFK I is inactivated through increased expression of PFK II.

How is the expression of pfkA regulated in uropathogenic E. coli strains?

In uropathogenic Escherichia coli (UPEC) strains, the regulation of pfkA demonstrates intriguing patterns that differ from what might be expected based on its central metabolic role. During urinary tract infection (UTI), UPEC strains display remarkable metabolic flexibility to adapt to the host environment . Surprisingly, UPEC strains lacking the major phosphofructokinase (pfkA) show no fitness defect during UTI, indicating compensatory mechanisms at work .

This unexpected finding is explained by the presence of the isozyme PFK II encoded by pfkB. The functional redundancy between these two enzymes in the infection context suggests that UPEC has evolved metabolic plasticity to thrive in the urinary tract environment. Even more surprisingly, strains lacking both phosphofructokinase-encoding genes (pfkA pfkB double mutant) outcompete the parental strain in the bladder . This counterintuitive finding suggests that redirecting carbon flux away from glycolysis may actually be advantageous during certain stages of infection.

These observations indicate that the regulation of central carbon metabolism in UPEC during infection is complex and context-dependent. The findings also suggest that the expression of pfkA might be actively suppressed under certain conditions to provide a fitness advantage. This phenomenon of suppressing latent enzymes appears to be a broader strategy employed by UPEC, as similar patterns are observed with pyruvate kinase genes, where loss of pykF results in a colonization defect while loss of pykA provides a fitness advantage . These findings challenge conventional assumptions about the importance of glycolytic enzymes during infection and highlight the sophisticated metabolic adaptation strategies employed by pathogenic bacteria.

How can factorial design be applied to optimize recombinant pfkA expression?

Factorial design offers a powerful experimental approach for optimizing recombinant pfkA expression by systematically evaluating multiple variables and their interactions simultaneously. This methodology is more efficient than traditional one-factor-at-a-time approaches and can reveal important interaction effects that might otherwise be missed2.

In a factorial design for pfkA expression optimization, researchers would first identify key factors affecting protein expression, such as:

• Expression temperature (e.g., 18°C, 25°C, 37°C)

• Inducer concentration (e.g., 0.1 mM, 0.5 mM, 1.0 mM IPTG)

• Media composition (e.g., LB, TB, defined media)

• Induction timing (early, mid, or late log phase)

For a simple 2² factorial design exploring temperature and inducer concentration, experiments would be conducted for all four combinations of the two variables at their high and low levels2. For example:

After conducting experiments under each condition, researchers would analyze the results to determine:

• The main effect of temperature: [(Yield₃+Yield₄)/2 - (Yield₁+Yield₂)/2]

• The main effect of IPTG concentration: [(Yield₂+Yield₄)/2 - (Yield₁+Yield₃)/2]

• The interaction effect: [(Yield₁+Yield₄)/2 - (Yield₂+Yield₃)/2]

Statistical methods like ANOVA would then be used to determine the significance of these effects2. This approach allows researchers to identify optimal conditions for pfkA expression while gaining insights into how different factors interact. For instance, factorial design might reveal that lower temperatures improve soluble pfkA yield but only when combined with lower inducer concentrations—an interaction that would not be evident when changing one factor at a time.

What purification strategies are most effective for obtaining active recombinant pfkA?

Purifying active recombinant pfkA requires careful consideration of the enzyme's structural and biochemical properties. The most effective purification strategies maintain the enzyme's quaternary structure and activity throughout the process. Based on approaches used for similar phosphofructokinases, the following multi-step strategy is recommended:

For His-tagged recombinant pfkA:

• Cell lysis under gentle conditions: Sonication or French press in a buffer containing 50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, 1-5 mM DTT, 5-10% glycerol, and protease inhibitors . Maintaining reducing conditions is crucial for preserving enzymatic activity.

• Immobilized Metal Affinity Chromatography (IMAC): The initial capture step using Ni-NTA or Co-based resins, with:

  • Binding buffer: Same as lysis buffer with 10-20 mM imidazole

  • Washing: Gradual increase to 50 mM imidazole to remove non-specific binding

  • Elution: 250-300 mM imidazole in the same buffer base

• Size Exclusion Chromatography: Critical for isolating properly folded tetrameric pfkA and removing aggregates or improperly assembled forms . This step also allows buffer exchange to remove imidazole, which can interfere with activity assays.

• Optional Ion Exchange Chromatography: As a polishing step if higher purity is required, typically using anion exchange (Q-Sepharose) at pH 8.0.

For native (untagged) pfkA, alternative approaches include:

• Ammonium sulfate fractionation: Typically, PFK precipitates between 35-55% saturation

• Ion exchange chromatography: Using DEAE or Q-Sepharose columns

• ATP-agarose affinity chromatography: Leveraging the enzyme's affinity for its substrate

Throughout all purification steps, it's essential to maintain the enzyme at 4°C and include glycerol (5-10%) and reducing agents (DTT or β-mercaptoethanol) in all buffers to preserve the quaternary structure and activity . Quality control should include SDS-PAGE for purity assessment, native PAGE to confirm tetrameric structure, and activity assays to verify enzymatic function. This approach typically yields highly pure and active pfkA suitable for structural and functional studies.

What is known about the quaternary structure of E. coli pfkA?

E. coli PFK I (encoded by pfkA) functions as a homotetramer in its active form, with four identical subunits each having a molecular weight of approximately 34,000 Da . The quaternary structure is essential for both catalytic activity and allosteric regulation. The tetrameric assembly has D2 symmetry and is arranged as a dimer of dimers, with each subunit interfacing with three other subunits.

The active sites of PFK are formed at the interfaces between subunits, with each tetramer containing four active sites for substrate binding and catalysis. Additionally, there are four allosteric effector sites that bind regulatory molecules such as ADP (activator) and phosphoenolpyruvate (inhibitor) . This arrangement enables the cooperative binding of substrate and the transmission of conformational changes throughout the tetramer when effectors bind.

The quaternary structure facilitates the allosteric behavior of PFK I, allowing it to transition between different conformational states (often described as R (relaxed) and T (tense) states) in response to substrate binding and the presence of allosteric effectors. These transitions mediate the cooperative behavior observed with fructose-6-phosphate binding . The tetramer is stabilized by extensive inter-subunit contacts, and dissociation into monomers results in complete loss of enzymatic activity.

This quaternary organization distinguishes PFK I from PFK II (encoded by pfkB), which, while also tetrameric, has different regulatory properties and slightly larger subunits (36,000 Da) . The quaternary structure of PFK I is critical for its function as a key control point in glycolysis, allowing for precise regulation of glycolytic flux in response to cellular energy status.

How does the structure of E. coli pfkA compare to phosphofructokinase enzymes in other organisms?

The structure of E. coli pfkA-encoded phosphofructokinase (PFK I) shows both conservation and divergence when compared to PFKs from other organisms, reflecting evolutionary adaptations to different metabolic requirements.

The most striking structural difference is observed between bacterial and eukaryotic PFKs. Eukaryotic PFK monomers are approximately twice the size of bacterial counterparts (~85 kDa vs ~34 kDa), suggesting an evolutionary gene duplication and fusion event . This size difference reflects the more complex regulatory requirements in eukaryotic cells. Human PFKs, for example, exist in three isoforms (PFK-M, PFK-L, and PFK-P) that can form homo- or heterotetramers with distinct regulatory properties .

In humans, the three PFK isoforms (muscle, liver, and platelet types) show tissue-specific expression patterns and different kinetic properties . These isoforms respond to additional regulators not found in bacterial systems, such as fructose-2,6-bisphosphate, which is a potent activator of eukaryotic PFKs but has no effect on bacterial enzymes like E. coli pfkA.

Despite these structural differences, the catalytic mechanism is largely conserved across species, with the active site architecture being more preserved than regulatory sites. This conservation reflects the essential nature of the phosphoryl transfer reaction in glycolysis, while the diversity in regulatory mechanisms highlights how PFK has evolved to meet different metabolic control requirements across organisms.

What kinetic properties distinguish E. coli pfkA from pfkB?

The kinetic properties of E. coli PFK I (encoded by pfkA) and PFK II (encoded by pfkB) reveal fundamental differences in their regulatory mechanisms and metabolic roles. These distinct properties explain why PFK I serves as the main glycolytic enzyme under normal conditions, while PFK II plays a supplementary role.

The most significant kinetic difference is in their response to substrates and regulators:

PFK I exhibits allosteric properties characteristic of a metabolic control point, including cooperative binding of fructose-6-phosphate with a Hill coefficient of approximately 3-4 . This cooperativity allows for a sensitive response to small changes in substrate concentration. In contrast, PFK II shows hyperbolic kinetics typical of non-regulatory enzymes .

Another key distinction is in their response to metabolic regulators. PFK I is strongly inhibited by phosphoenolpyruvate (an indicator of high energy status) and activated by ADP (signaling low energy status), providing a mechanism for adjusting glycolytic flux according to cellular energy requirements . PFK II lacks these regulatory responses, functioning as a constitutive enzyme regardless of energy status.

These kinetic differences explain why PFK I serves as the main regulatory enzyme under normal conditions, while PFK II provides a metabolic backup that becomes important when PFK I is inhibited or mutated, as observed in strains carrying the pfkB1 suppressor mutation . The complementary kinetic properties of these isozymes contribute to the metabolic flexibility of E. coli, allowing glycolysis to continue under various conditions.

How do mutations in pfkA affect E. coli metabolism and fitness?

In response to pfkA mutations, E. coli activates compensatory mechanisms, most notably the upregulation of pfkB expression. This is particularly evident in strains carrying the pfkB1 mutation, which acts as a suppressor of pfkA mutations by increasing PFK II levels . This genetic suppression allows E. coli to maintain glycolytic flux despite the absence of the major phosphofructokinase.

Interestingly, the fitness effects of pfkA mutations can vary dramatically depending on the environment. In uropathogenic E. coli (UPEC) strains during urinary tract infection, strains lacking pfkA show no fitness defect . Even more surprisingly, strains lacking both phosphofructokinase-encoding genes (pfkA pfkB double mutant) outcompete the parental strain in the bladder . This counterintuitive finding suggests that redirecting carbon flux away from glycolysis may be advantageous in certain infection contexts.

The metabolic rewiring that occurs in pfkA mutants likely involves:

• Increased pentose phosphate pathway flux

• Enhanced gluconeogenesis

• Altered TCA cycle activity

• Accumulation of upstream metabolites, particularly glucose-6-phosphate and fructose-6-phosphate

These adaptations reflect the integrated nature of central carbon metabolism and demonstrate how bacteria can adjust to severe metabolic perturbations through various compensatory mechanisms. The observation that pfkA mutations can sometimes enhance fitness in specific environments challenges conventional assumptions about the importance of glycolysis and highlights the complexity of bacterial metabolic adaptation strategies.

What role does pfkA play in bacterial pathogenesis and host adaptation?

The role of pfkA in bacterial pathogenesis, particularly in uropathogenic E. coli (UPEC), reveals sophisticated metabolic adaptation strategies during infection. UPEC strains demonstrate remarkable metabolic flexibility in the urinary tract environment, where the regulation of central carbon metabolism differs significantly from laboratory conditions .

Surprisingly, UPEC strains lacking the major phosphofructokinase (ΔpfkA) show no fitness defect during urinary tract infection (UTI) . This unexpected finding is explained by the presence of the isozyme encoded by pfkB, which compensates for the loss of the major phosphofructokinase. Even more remarkable, strains lacking both phosphofructokinases (ΔpfkA pfkB double mutant) actually outcompete the parental strain in the bladder during infection . This suggests that redirecting carbon flux away from glycolysis may be advantageous in the bladder environment.

Similar patterns are observed with pyruvate kinase genes in UPEC, where loss of pykF (encoding Pyk I) results in a colonization defect, while loss of pykA (encoding Pyk II) provides a fitness advantage . The pykA pykF double mutant performs similarly to the wild-type strain during infection, suggesting complex interactions between these isozymes during pathogenesis.

These findings indicate that UPEC employs a strategy of suppressing certain "latent" enzymes to optimize fitness in the host environment . This metabolic flexibility allows UPEC to adapt to the unique nutritional landscape of the urinary tract, where conventional glycolysis may not be the optimal metabolic strategy.

The implications for pathogenesis and treatment are significant. Understanding how pathogens regulate metabolism in vivo provides insights for developing new antimicrobial strategies. Rather than targeting the most active metabolic enzymes, effective treatments might instead focus on the backup systems that become essential during infection. This research highlights the importance of studying bacterial metabolism in relevant host environments rather than extrapolating from laboratory conditions.

How can structural information about pfkA inform inhibitor design for antimicrobial development?

Structural knowledge of E. coli pfkA provides valuable insights for rational inhibitor design, potentially leading to novel antimicrobials targeting this essential metabolic enzyme. Several structural features of pfkA make it an attractive target for inhibitor development:

• Active site architecture: The active site of pfkA is formed at the interface between subunits, creating a unique environment that differs from mammalian PFK counterparts . These structural differences can be exploited to develop inhibitors with selectivity for bacterial PFK over human isoforms, reducing potential side effects.

• Allosteric binding sites: Beyond the active site, pfkA contains allosteric regulatory sites that bind modulators like phosphoenolpyruvate (inhibitor) and ADP (activator) . These sites offer alternative targeting opportunities, potentially allowing for inhibitors that lock the enzyme in an inactive conformation.

• Quaternary structure interfaces: The tetrameric assembly of pfkA is essential for its function . Molecules that disrupt tetramer formation or stability could serve as effective inhibitors without directly competing with substrates.

• Bacterial-specific regulatory mechanisms: The unique regulatory properties of bacterial PFK, which responds to different allosteric effectors than mammalian enzymes, provides opportunities for selective targeting .

In developing inhibitors, researchers might pursue several strategies:

• Substrate analogs that compete for the active site but cannot be phosphorylated

• Allosteric inhibitors that bind to regulatory sites and stabilize inactive conformations

• Interface disruptors that prevent proper tetramer assembly

• Covalent modifiers that target accessible cysteine residues unique to bacterial PFK

Successful inhibitor design would also need to consider various challenges:

• Delivery across the bacterial cell wall and membrane

• Potential for resistance development

• Metabolic bypass through alternative pathways or isozymes (like PFK II)

What are common challenges in expressing recombinant pfkA and how can they be addressed?

Researchers working with recombinant E. coli pfkA often encounter several challenges during expression and purification that require specific troubleshooting approaches:

• Solubility issues: PFK I can form inclusion bodies, especially at high expression levels. This can be addressed by:

  • Lowering induction temperature (16-25°C)

  • Reducing inducer concentration

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

  • Using solubility-enhancing fusion tags (MBP, SUMO)

  • Optimizing media composition with osmolytes or mild solubilizers

• Maintaining quaternary structure: Loss of tetrameric structure during purification can lead to inactive enzyme . Solutions include:

  • Including stabilizing agents in buffers (glycerol, low concentrations of substrates)

  • Avoiding harsh purification conditions

  • Using gentle lysis methods

  • Employing native PAGE to monitor quaternary state

  • Performing size exclusion chromatography to isolate properly formed tetramers

• Enzymatic activity loss: Purified protein may show low or no activity due to various factors. Address this by:

  • Maintaining reducing environment (DTT or β-mercaptoethanol)

  • Including Mg²⁺ in activity assays (essential cofactor)

  • Checking pH (activity optimal at pH 7.5-8.0)

  • Verifying tetrameric assembly

  • Testing for co-purifying inhibitors

• Proteolytic degradation: Prevents obtaining intact protein. Solutions include:

  • Using protease-deficient expression strains

  • Adding protease inhibitors during all purification steps

  • Reducing purification time and maintaining cold temperature

  • Adding stabilizing ligands during purification

• Co-purification of host proteins: E. coli host proteins contaminating purified pfkA. Address by:

  • Employing multiple orthogonal purification steps

  • Using more stringent washing conditions during affinity chromatography

  • Considering alternative tagging strategies

  • Implementing negative selection steps to remove common contaminants

By anticipating and addressing these common challenges, researchers can improve the efficiency and reproducibility of recombinant pfkA expression and purification, obtaining high-quality enzyme for structural and functional studies.

What controls should be included in experimental designs studying pfkA function?

Robust experimental design for studying pfkA function requires comprehensive controls to ensure reliable and interpretable results. These controls should address enzyme quality, assay conditions, and biological context:

• Enzyme-specific controls:

  • Enzyme blank: Complete reaction mixture without pfkA to measure background rate

  • Heat-inactivated enzyme: pfkA sample heated to 95°C for 10 minutes to denature the enzyme

  • Concentration-dependent activity: Serial dilutions of pfkA to confirm linearity of enzyme activity

  • Wild-type vs. mutant comparison: Include wild-type pfkA alongside any mutant variants

• Substrate and cofactor controls:

  • Substrate blanks: Omit each substrate individually (F6P, ATP) to detect non-specific activities

  • Substrate quality control: Verify the purity and concentration of F6P and ATP stocks

  • Magnesium dependence: Confirm the requirement for Mg²⁺ by testing activity in its absence

• Assay validation controls:

  • Coupling enzyme verification: For coupled assays, directly test the activity of coupling enzymes

  • Alternative assay method: Validate results using an orthogonal activity assay where possible

  • Temperature and pH controls: Verify assay conditions are optimal for pfkA activity

• Regulatory mechanism controls:

  • Allosteric effector tests: Include known activators (ADP) and inhibitors (PEP) to verify regulatory function

  • Cooperativity verification: Perform substrate-velocity curves to confirm sigmoidal kinetics for F6P

• In vivo functional controls:

  • Complementation tests: Verify that recombinant pfkA can restore growth in pfkA-deficient strains

  • Isozyme comparison: Compare with pfkB to distinguish isozyme-specific effects

  • Growth condition variations: Test function under different carbon sources and stress conditions

• In silico controls:

  • Sequence verification: Confirm the recombinant construct matches the expected sequence

  • Structural modeling: Use homology modeling to predict effects of mutations or modifications

• Experimental design controls:

  • Factorial design: When optimizing expression or studying multiple variables, use factorial design to identify interactions between factors2

  • Technical and biological replicates: Include sufficient replication for statistical validation

  • Appropriate statistical tests: Apply suitable statistical methods for data analysis

By incorporating these controls, researchers can ensure the validity of their findings about pfkA function, distinguish true effects from artifacts, and generate reproducible results that advance our understanding of this important metabolic enzyme and its role in bacterial physiology.

How can researchers distinguish between the activities of pfkA and pfkB in experimental systems?

Distinguishing between the activities of PFK I (encoded by pfkA) and PFK II (encoded by pfkB) in experimental systems is crucial for understanding their specific roles and contributions to bacterial metabolism. Several approaches can be employed to differentiate between these isozymes:

• Genetic approaches:

  • Use single-gene knockout strains (ΔpfkA or ΔpfkB) to study each isozyme in isolation

  • Create double knockout strains (ΔpfkA ΔpfkB) complemented with either pfkA or pfkB for controlled expression

  • Employ regulated expression systems to tune the levels of each isozyme independently

• Kinetic differentiation:

  • Exploit the differential response to allosteric regulators: PFK I is inhibited by phosphoenolpyruvate and activated by ADP, while PFK II shows no response to these modulators

  • Measure cooperativity with fructose-6-phosphate: PFK I exhibits sigmoidal kinetics (Hill coefficient >1), while PFK II shows hyperbolic kinetics

  • Test for inhibition by fructose-1,6-bisphosphate, which affects PFK II but not PFK I

• Substrate specificity:

  • Assess activity with alternative substrates: PFK II can use tagatose-6-phosphate as a substrate, while PFK I cannot

  • Compare the kinetic parameters (Km, Vmax) for the primary substrates, as they differ between the isozymes

• Immunological differentiation:

  • Use isozyme-specific antibodies for detection and quantification, as PFK I and PFK II show no immunological cross-reactivity

  • Employ immunoprecipitation to selectively remove one isozyme from mixed samples

• Physical separation:

  • Use chromatographic techniques (ion exchange, size exclusion) to separate the isozymes based on their differences in surface charge and molecular weight

  • Apply native gel electrophoresis to distinguish between the enzymes based on mobility differences

• Mass spectrometry approaches:

  • Identify and quantify isozyme-specific peptides in complex samples using targeted proteomics

  • Monitor post-translational modifications that might differentially affect the isozymes

• Thermal stability differentiation:

  • Evaluate thermal inactivation profiles, as the isozymes likely have different thermal stabilities

  • Use differential scanning fluorimetry to compare unfolding temperatures

By combining these approaches, researchers can effectively distinguish between pfkA and pfkB activities in various experimental contexts, from purified enzyme systems to complex cellular environments. This differentiation is essential for understanding how these isozymes contribute to metabolic flexibility and adaptation in different growth conditions and during host infection .