Recombinant Staphylococcus aureus 6-phosphofructokinase (pfkA)

What is Staphylococcus aureus 6-phosphofructokinase (pfkA)?

pfkA is a key glycolytic enzyme that catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, representing the first committed step of glycolysis in S. aureus. This ATP-dependent enzyme (EC 2.7.1.11) is also known as phosphofructokinase or phosphohexokinase. The recombinant form refers to the protein expressed in laboratory systems for research purposes, typically derived from S. aureus strain USA300/TCH1516 with UniProt accession number A8Z2L4 . The enzyme plays a critical role in central carbon metabolism and energetics of S. aureus, making it relevant for understanding bacterial physiology and potential antimicrobial targets.

How does pfkA function in S. aureus metabolism?

In S. aureus, pfkA catalyzes a crucial step in central carbon metabolism, converting fructose-6-phosphate to fructose-1,6-bisphosphate while consuming ATP. This reaction represents the first committed step of glycolysis and is often rate-limiting, making it a key control point for regulating cellular energy production. Recent transcriptomics and metabolomics analyses have revealed that pfkA expression is regulated by factors such as small RNA SprC, which affects various metabolic pathways in S. aureus . Furthermore, pfkA functions within a broader metabolic network, interacting with other glycolytic enzymes and transporters, including the carbohydrate phosphotransferase system (PTS) . This positioning makes pfkA activity essential for bacterial growth, particularly under conditions where glucose is the primary carbon source.

What are the optimal storage conditions for recombinant S. aureus pfkA?

For optimal stability and activity maintenance of recombinant S. aureus pfkA, the following storage conditions are recommended:

• Short-term storage: Store at -20°C

• Extended storage: Store at -20°C or preferably -80°C

• Working aliquots: Can be stored at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they may compromise protein integrity and activity

For liquid formulations, the typical shelf life is approximately 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for up to 12 months at these temperatures . These storage recommendations aim to preserve the structural integrity and enzymatic activity of pfkA for experimental use.

How should recombinant S. aureus pfkA be reconstituted for experimental use?

For optimal reconstitution of recombinant S. aureus pfkA:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a final concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) to enhance stability for long-term storage

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store aliquots at -20°C or -80°C for extended preservation

For experimental reproducibility, it is critical to document the reconstitution buffer composition, as buffer components can influence enzyme activity. When preparing working solutions, consider the ionic strength requirements for optimal pfkA activity and the compatibility with downstream experimental applications.

What assays can be used to measure S. aureus pfkA enzymatic activity?

Several assay methods can be employed to measure pfkA activity in S. aureus:

• Coupled spectrophotometric assay: This commonly used method links pfkA activity to NADH oxidation through coupling enzymes like aldolase, triosephosphate isomerase, and α-glycerophosphate dehydrogenase. The decrease in NADH absorbance at 340 nm correlates with pfkA activity .

• Direct product quantification: Measuring the formation of fructose-1,6-bisphosphate using chromatographic methods such as HPLC or LC-MS.

• ATP consumption assay: Monitoring the consumption of ATP using luciferase-based assays or other ATP detection methods.

For kinetic characterization, researchers typically measure initial reaction velocities at varying concentrations of substrates (fructose-6-phosphate and ATP). The data can be fitted to appropriate enzyme kinetic models, such as the Michaelis-Menten equation, Hill equation for sigmoidal kinetics, or Haldane's equation when substrate inhibition is observed . These methodological approaches enable quantitative assessment of pfkA activity under different experimental conditions.

How is pfkA activity regulated in S. aureus?

The regulation of pfkA in S. aureus involves multiple mechanisms:

• Allosteric regulation: By analogy with other bacterial phosphofructokinases, S. aureus pfkA activity is likely modulated by metabolic intermediates. Studies of phosphofructokinases in other bacteria show that these enzymes can be inhibited by excess substrate (ATP and fructose-6-phosphate) and reaction products (ADP and fructose-1,6-bisphosphate) .

• Transcriptional regulation: Small RNA SprC has been shown to affect pfkA expression, though with inconsistent effects across different studies . This regulatory RNA appears to influence various metabolic pathways in S. aureus.

• Environmental regulation: Metabolic shifts in response to oxygen availability, nutrient conditions, and stress responses may indirectly affect pfkA expression and activity.

• Post-translational modification: Research indicates that pfkA can be targeted by antimicrobial agents like silver ions (Ag+), which can bind to the protein and potentially disrupt its function .

These regulatory mechanisms highlight the integration of pfkA within the complex metabolic network of S. aureus, allowing the bacterium to adapt its energy metabolism to changing environmental conditions.

What is the role of pfkA in S. aureus virulence and pathogenesis?

While direct evidence linking pfkA to S. aureus virulence is limited in the provided sources, several indirect connections suggest its importance in pathogenesis:

• Central metabolic role: As a key glycolytic enzyme, pfkA contributes to energy production necessary for bacterial growth and survival during infection.

• Relation to virulence regulators: Transcriptomic studies reveal that small RNA SprC, which affects pfkA expression, also modulates known virulence factors like LukD and LukE toxins .

• Biofilm formation: Metabolic enzymes including pfkA may indirectly influence biofilm formation, an important virulence trait of S. aureus. Studies indicate that glycolytic activity and ATP production affect biofilm development .

• Antimicrobial target: pfkA has been identified as a target for silver ions, suggesting its importance in bacterial survival under antimicrobial stress .

These connections highlight that while pfkA's primary role is metabolic, its function has implications for virulence-associated processes in S. aureus, making it relevant for understanding pathogen biology.

How does silver (Ag+) targeting of pfkA contribute to antimicrobial effects against S. aureus?

Silver ions (Ag+) have been identified as potential antimicrobials against antibiotic-resistant S. aureus, with pfkA among the key molecular targets. Research indicates that:

• Ag+ can directly bind to pfkA along with other glycolytic enzymes (enolase, pyruvate dehydrogenase E1 component subunit beta, and lactate dehydrogenase) .

• This binding appears to functionally disrupt glycolysis, which bioinformatics analysis identified as the most significantly enriched biological pathway affected by silver treatment .

• By targeting multiple enzymes in this critical metabolic pathway, including pfkA, silver exerts a multi-target mode of action that may help overcome bacterial resistance mechanisms.

• The essential role of pfkA in energy metabolism makes its inhibition particularly detrimental to bacterial survival and growth.

This research direction highlights the potential of targeting metabolic enzymes like pfkA as an alternative strategy to combat antibiotic-resistant S. aureus infections, potentially opening new avenues for antimicrobial development.

What differences exist between pfkA and pfkB isoenzymes in bacteria?

While the provided sources focus primarily on S. aureus pfkA rather than comparing pfkA and pfkB in this organism specifically, research on Mycobacterium tuberculosis phosphofructokinases reveals significant functional differences between these isoenzymes that may have relevance for understanding their roles in other bacteria:

These differences suggest complementary roles for these isoenzymes, with pfkA supporting primary glycolytic flux under optimal conditions, while pfkB may maintain glycolytic function under stress conditions that inhibit pfkA activity . Whether similar functional differentiation exists in S. aureus warrants further investigation.

How can heterologous expression systems be optimized for studying S. aureus pfkA?

Heterologous expression of S. aureus pfkA requires careful consideration of several factors:

• Vector selection: Appropriate expression vectors with compatible promoters and selection markers are critical. Research has utilized vectors like pRL with specific modifications for pfkA expression .

• Host selection: Expression hosts must be chosen based on research objectives. While E. coli is commonly used for protein production, other systems may be more suitable for studying functional aspects or interactions specific to gram-positive bacteria.

• Codon optimization: Adjusting the coding sequence to match the codon usage bias of the expression host can improve translation efficiency and protein yields.

• Tag incorporation: Addition of affinity tags (like FLAG-tag) facilitates protein purification and detection, though careful placement is needed to avoid interfering with enzyme function .

• Co-expression considerations: When studying pfkA in relation to other glycolytic enzymes, co-expression vectors containing multiple genes (e.g., pfkA with tpiA and fbaA) can be engineered, though recombination between similar sequences must be minimized .

• Verification methods: Expression should be verified through methods like Western blotting, enzyme activity assays, and mass spectrometry to confirm proper folding and function of the recombinant protein.

These approaches enable researchers to produce active S. aureus pfkA for biochemical characterization, structural studies, and investigation of potential inhibitors with antimicrobial properties.

What contradictions exist in current research regarding pfkA regulation in S. aureus?

Current research reveals some inconsistencies regarding pfkA regulation in S. aureus:

• The role of small RNA SprC in regulating pfkA expression shows contradictory results. While transcriptomic analysis identified pfkA as potentially regulated by SprC, the exact direction and mechanism of this regulation remains unclear, with inconsistent results reported across studies .

• The specific allosteric regulators of S. aureus pfkA have not been definitively characterized. While research on phosphofructokinases in other bacteria suggests regulation by metabolites like fructose-2,6-bisphosphate, citrate, and phosphoenolpyruvate, the specific regulatory profile of S. aureus pfkA requires further investigation .

• The relative importance of transcriptional versus post-translational regulation of pfkA activity in S. aureus under different environmental conditions remains to be fully elucidated.

These contradictions highlight the need for further research specifically focused on S. aureus pfkA regulation in various physiological and stress conditions.

How can pfkA be targeted for antimicrobial development against S. aureus?

Developing antimicrobials targeting pfkA presents both opportunities and challenges:

• Target validation: As a key glycolytic enzyme, pfkA represents a potential vulnerability in S. aureus metabolism. The identification of pfkA as a target for silver antimicrobials provides preliminary validation of this approach .

• Inhibitor design strategies:

  • Structure-based design targeting the unique features of bacterial pfkA compared to human phosphofructokinase

  • Allosteric inhibitors that exploit the regulatory mechanisms of bacterial pfkA

  • Covalent modifiers that interact with specific residues in S. aureus pfkA

• Combination approaches: Targeting pfkA alongside other glycolytic enzymes might create synergistic effects, similar to the multi-target action observed with silver .

• Delivery challenges: Developing compounds that can effectively penetrate the S. aureus cell wall while maintaining selectivity for bacterial versus human enzymes.

• Resistance development: Evaluating the potential for resistance development through mutations in pfkA or metabolic bypasses.

This research direction could lead to novel therapeutic approaches against antibiotic-resistant S. aureus strains, particularly if inhibitors can be designed with sufficient selectivity for bacterial versus human phosphofructokinases.