Recombinant Clostridium novyi 6-phosphofructokinase (pfkA)

What is the molecular function of 6-phosphofructokinase in C. novyi metabolism?

6-phosphofructokinase (pfkA) in C. novyi functions as an ATP-dependent enzyme (EC 2.7.1.11) that catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, a rate-limiting step in glycolysis. This reaction is critical for central carbon metabolism, enabling the bacterium to process glucose for energy production .

The reaction catalyzed by pfkA can be represented as:
$ATP + D-fructose 6-phosphate → ADP + D-fructose 1,6-bisphosphate$

In the context of C. novyi metabolism, pfkA activity influences multiple downstream metabolic pathways, including:

• Glycolysis for ATP generation

• Carbon flux distribution between glycolysis and pentose phosphate pathway

• Metabolite availability for biosynthetic processes

How does C. novyi pfkA differ structurally from other bacterial phosphofructokinases?

While the search results don't provide complete structural information specific to C. novyi pfkA, general properties can be inferred from related bacterial phosphofructokinases and available information:

• C. novyi pfkA is annotated as NT01CX_1297 in the genome sequence

• The enzyme belongs to the ATP-dependent phosphofructokinase family

• Unlike mammalian PFK, which exists as tetramers with complex allosteric regulation, bacterial PFKs typically function as homotetramers with somewhat simpler regulatory mechanisms

• C. novyi pfkA likely contains AMP/ADP allosteric activation sites, similar to phosphofructokinase liver type (PFKL) in humans

Further structural studies using X-ray crystallography would be valuable to determine specific structural features unique to C. novyi pfkA.

Expression and Purification Methodologies

Based on protocols used for phosphofructokinase assays in related research, the following conditions would be appropriate for measuring C. novyi pfkA activity:

A coupled enzymatic assay can be performed at room temperature (approximately 25°C) in a buffer containing:

• 100 mM Tris-HCl, pH 8.2

• 1 mM ATP

• 0.2 mM β-NADH

• 1 mM fructose-6-phosphate (F6P)

• 1 mM NH₄Cl

• 0.01% Triton X-100

• Coupling enzymes: 0.83 U aldolase, 0.42 U triosephosphate isomerase, and 0.42 U glycerophosphate dehydrogenase

The reaction progress can be monitored by measuring absorbance at 340 nm, where one unit of pfkA activity is defined as the amount required to convert 1.0 μmole of ATP and D-fructose 6-phosphate to ADP and fructose 1,6-bisphosphate per minute at pH 8.2 .

How can manipulation of pfkA expression be used to redirect metabolic flux in C. novyi?

Controlled manipulation of pfkA expression levels provides a powerful approach for redirecting metabolic flux in C. novyi:

• Upregulation of pfkA: Increases glycolytic flux, potentially enhancing ATP production and growth rate, but may reduce flux through alternative pathways like the pentose phosphate pathway

• Downregulation or knockdown of pfkA: Can redirect glucose-6-phosphate toward alternative pathways, including:

  • The pentose phosphate pathway, increasing NADPH production

  • Heterologous biosynthetic pathways that utilize glucose-6-phosphate as a precursor

• Dynamic control strategies: Post-translational control of pfkA through modified SsrA tags allows for temporal control of enzyme levels, enabling a switch between growth-optimized and production-optimized metabolic states

Research with E. coli has demonstrated that controlled degradation of phosphofructokinase-I (Pfk-I) increased the glucose-6-phosphate pool available for conversion into myo-inositol, achieving a two-fold improvement in yield and titers . Similar approaches could be applied to C. novyi for metabolic engineering purposes.

What challenges exist in using pfkA manipulation for metabolic engineering in obligate anaerobes like C. novyi?

Several significant challenges must be addressed when manipulating pfkA in obligate anaerobes like C. novyi:

• Oxygen sensitivity: Working with C. novyi requires strict anaerobic conditions, complicating experimental procedures. Recent methodological advances now permit some benchtop work with this obligate micro-anaerobe .

• Metabolic balance: PfkA manipulations can have unexpected consequences due to the interconnected nature of metabolic networks. In a study with Zymomonas mobilis, expression of heterologous Pfk-I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene .

• Redox balance: Altering glycolytic flux can disrupt NAD⁺/NADH ratios, potentially affecting numerous cellular processes in anaerobic metabolism.

• Genetic tool limitations: While CRISPR/Cas9 methods have been developed for C. novyi , genetic manipulation of obligate anaerobes remains technically challenging compared to model organisms.

• Metabolic context effects: Native metabolite concentrations can cause heterologous reactions to operate in unexpected directions, as observed in Zymomonas mobilis .

A potential solution involves developing specialized cultivation methods such as sporulation media containing 500 mg Na₂HPO₄, 3 g peptone, 0.05 g L-cysteine, and 1 g maltose per 100 mL, with pH adjusted to 7.5 and supplemented with dried cooked meat particles at 0.5% w/v .

How does pfkA activity relate to the virulence mechanisms of C. novyi?

While pfkA itself is not directly identified as a virulence factor in the provided research, there are potential connections between central metabolism and pathogenicity:

• Energy production for toxin synthesis: As a key glycolytic enzyme, pfkA contributes to ATP generation needed for various cellular processes, including the production of virulence factors like the alpha-toxin (TcnA) .

• Metabolic adaptation: PfkA activity may influence C. novyi's ability to adapt to different host environments, particularly hypoxic conditions found in necrotic tissues.

• Indirect effects on virulence regulation: Metabolic shifts can influence global gene regulation, potentially affecting expression of virulence genes.

The alpha-toxin of C. novyi is well-characterized as the primary virulence factor, modifying small GTPases by N-acetylglucosamination and causing cell death . Alpha-toxin activity results in alterations to 23 common signaling pathways, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases .

What role might pfkA play in C. novyi-NT's ability to target hypoxic tumor environments?

C. novyi-NT (a non-toxic strain with the alpha-toxin gene eliminated) has demonstrated selective colonization of hypoxic tumor environments . The potential roles of pfkA in this tumor-targeting capability include:

• Metabolic adaptation to hypoxia: PfkA is crucial for glycolysis, which becomes the primary energy-generating pathway under the hypoxic conditions found in tumor cores. The enzyme's activity may be optimized for function in low-oxygen environments.

• Carbon flux distribution: PfkA regulation could influence the balance between glycolysis and the pentose phosphate pathway, affecting NADPH production. This balance is particularly relevant as NADPH from the pentose phosphate pathway fuels NADPH oxidase (NOX2) to produce reactive oxygen species in neutrophils .

• Sporulation and germination: PfkA activity may influence energy availability for sporulation and subsequent germination processes that are critical for C. novyi-NT therapeutic action. C. novyi spores selectively germinate in the hypoxic tumor environment .

Research has shown that C. novyi-NT can be modified using CRISPR/Cas9 to express tumor-targeting peptides like RGD on spore coat proteins, enhancing tumor localization by binding to αvβ3 integrin commonly overexpressed on the epithelial tissue surrounding tumors .

How can CRISPR/Cas9 techniques be optimized for modifying pfkA in C. novyi?

CRISPR/Cas9 modification of C. novyi requires specialized approaches:

• Anaerobic protocols: All transformation and selection steps must be performed under strict anaerobic conditions in an anaerobic chamber or using specialized equipment to maintain viability .

• Delivery methods: Electroporation protocols must be optimized for C. novyi's cell wall characteristics. Research has successfully developed methods that allow for genetic customization of this obligate anaerobe .

• Guide RNA design: For targeting pfkA (NT01CX_1297), guide RNAs should be designed to target unique sequences with minimal off-target effects. Tools like PrimerExplorer 4.0 have been used for primer design in C. novyi .

• Homology-directed repair templates: For precise modifications of pfkA, HDR templates should include:

  • Homology arms (500-1000 bp) flanking the target site

  • Desired modifications to the pfkA sequence

  • Selection markers appropriate for anaerobic selection

• Screening methods: PCR verification using primers flanking the modified region, followed by sequencing to confirm the intended modifications.

Recent studies have demonstrated successful CRISPR/Cas9-mediated genetic modifications in C. novyi-NT, including insertion of genes encoding tumor-targeting peptides into spore coat protein promoter regions .

What methods are available for detecting intracellular levels of phosphorylated metabolites in C. novyi?

Measuring phosphorylated metabolites in C. novyi requires specialized techniques due to the organism's strict anaerobic nature:

• LC-MS/MS-based metabolomics:

  • Sample preparation must occur under anaerobic conditions to prevent metabolic shifts

  • Rapid quenching of metabolism using cold methanol (-40°C) or similar methods

  • Extraction using chloroform/methanol/water systems

  • Analysis using HILIC (Hydrophilic Interaction Liquid Chromatography) coupled to mass spectrometry

  • Targeted multiple reaction monitoring (MRM) for specific phosphorylated metabolites

• Enzymatic assays:
  For measuring specific metabolites like glucose-6-phosphate and fructose-6-phosphate, enzymatic cycling assays can be employed, similar to those described for E. coli studies . Working cultures should be inoculated to OD = 0.02 and incubated at appropriate temperature (typically 30-37°C for C. novyi).

• Electrochemical methods:
  Cyclic voltammetry methods have been developed for C. novyi detection and could potentially be adapted for metabolite analysis. This approach involves electrochemical ion bonding with positive dyes like methylene blue, resulting in measurable changes in oxidation peak current (ipA) and reduction peak current (ipC) .

• Isotope tracing:
  13C-labeled glucose can be used to trace carbon flux through glycolysis, with samples analyzed by GC-MS or LC-MS to determine isotope distribution in downstream metabolites.

For C. novyi grown under anaerobic conditions, special care must be taken throughout sample processing to prevent oxygen exposure that could alter metabolite profiles.

Comparative Analysis with Other Bacterial PFKs

Comparing C. novyi pfkA with human PFKL provides valuable insights into evolutionary adaptations and potential therapeutic targeting:

• Allosteric regulation:

  • Human PFKL is allosterically activated by AMP/ADP binding to specific sites

  • The small molecule NA-11 has been shown to activate PFKL by binding to the AMP/ADP allosteric activation site

  • Bacterial PFKs, including C. novyi pfkA, likely have distinct allosteric regulation mechanisms

• Isoform specificity:

  • Humans have three PFK isoforms (liver, platelet, and muscle types), while bacteria typically have a single PFK

  • NA-11 selectively activates PFKL but fails to agonize phosphofructokinase-1 platelet type (PFKP) or muscle type (PFKM)

  • This suggests potential for developing selective modulators of bacterial PFKs

• Metabolic impacts:

  • In neutrophils, PFKL activation by NA-11 dampens flux through the pentose phosphate pathway, reducing NADPH generation and inhibiting NOX2-dependent oxidative burst

  • Similar metabolic redirection could potentially be engineered in C. novyi by modulating pfkA activity

• Structural basis for regulation:

  • A high-resolution structure of human PFKL confirmed binding of NA-11 to the AMP/ADP allosteric activation site

  • Structural studies of C. novyi pfkA could identify unique regulatory sites that might be targetable for modulating its activity

These comparisons highlight the potential for developing selective modulators of bacterial versus human PFKs, which could have applications in both therapeutic development and metabolic engineering of C. novyi.

What are the main stability and storage considerations for recombinant C. novyi pfkA?

Based on available information about recombinant C. novyi pfkA and related proteins, the following stability and storage considerations are recommended:

• Storage temperature:

  • Store lyophilized recombinant protein at -20°C for long-term storage

  • For extended preservation, -80°C storage is preferable

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

• Freeze-thaw stability:

  • Avoid repeated freezing and thawing cycles as they can significantly reduce enzymatic activity

  • Prepare single-use aliquots when receiving or purifying the protein

• Buffer conditions for optimal stability:

  • pH 7.5-8.0 buffers (typically Tris-HCl or phosphate-based)

  • Addition of glycerol (10-20%) for freezing stability

  • Consider including reducing agents (1-5 mM DTT or 2-mercaptoethanol) to prevent oxidation of cysteine residues

• Lyophilization considerations:

  • Lyophilized formats tend to have better long-term stability

  • Reconstitution should use the recommended buffer to maintain activity

• Special considerations for C. novyi proteins:

  • As an obligate anaerobe product, oxidation during purification and storage may affect stability

  • Consider adding oxygen scavengers or performing manipulations under anaerobic conditions

Following these guidelines will help maintain enzyme activity and structural integrity for experimental applications.

How can researchers address common expression problems when producing recombinant C. novyi pfkA?

Researchers may encounter several challenges when expressing recombinant C. novyi pfkA. Here are methodological solutions to common problems:

• Poor expression levels:

  • Optimize codon usage for the expression host (E. coli, yeast, etc.)

  • Test multiple promoter systems (T7, tac, AOX1 for yeast)

  • Evaluate different growth temperatures (typically lower temperatures of 16-25°C improve solubility)

  • Consider fusion tags that enhance expression (MBP, SUMO, thioredoxin)

• Inclusion body formation in E. coli:

  • Decrease induction temperature to 16-20°C

  • Reduce inducer concentration

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Add solubility-enhancing agents to the media (sorbitol, glycine betaine)

  • Consider refolding protocols if inclusion bodies persist

• Loss of enzymatic activity:

  • Include cofactors in purification buffers (Mg²⁺ for pfkA)

  • Add stabilizing agents (glycerol, reducing agents)

  • Purify under anaerobic conditions to prevent oxidative damage

  • Test activity immediately after purification

• Toxicity to expression host:
  This is particularly relevant as expression of phosphofructokinase has been shown to cause growth inhibition in some cases :

  • Use tightly regulated expression systems

  • Try expression in specialized strains designed for toxic proteins

  • Use inducible degradation systems to control protein levels

• Degradation during purification:

  • Add protease inhibitors to all buffers

  • Work at lower temperatures (4°C)

  • Minimize purification time

  • Consider adding stabilizing excipients

• Mutations in expressed pfkA:
  Research has shown that expression of heterologous pfkA can result in accumulation of mutations , suggesting:

  • Sequence verify the expression construct before and after expression

  • Use specialized strains with lower mutation rates

  • Consider expressing in cell-free systems for problematic constructs

These methodological approaches should help overcome many of the common challenges in producing active recombinant C. novyi pfkA.