Recombinant Vibrio vulnificus 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB)

What is the role of 3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) in Vibrio vulnificus metabolism?

3-methyl-2-oxobutanoate hydroxymethyltransferase (panB) catalyzes a critical step in the pantothenate (vitamin B5) biosynthesis pathway in Vibrio vulnificus. This enzyme specifically converts 3-methyl-2-oxobutanoate (also known as α-ketoisovalerate) to 2-dehydropantoate by transferring a hydroxymethyl group. As a facultative pathogen that transitions between environmental and clinical settings, V. vulnificus relies on robust metabolic pathways to adapt to changing nutrient conditions. The panB enzyme represents an essential component of the bacterial metabolic network, as pantothenate is a precursor to coenzyme A (CoA), which participates in numerous cellular processes including fatty acid metabolism and energy production.

Studies investigating V. vulnificus genomic properties have demonstrated that metabolic versatility contributes significantly to its environmental adaptability and pathogenic potential . The panB gene is particularly conserved across Vibrio species, suggesting its evolutionary importance within the genus .

How does the genomic context of panB in Vibrio vulnificus compare to other Vibrio species?

In V. vulnificus, the panB gene is typically located within an operon structure related to pantothenate biosynthesis. Comparative genomic analysis reveals that while the gene is conserved across the Vibrionaceae family, its genomic context can vary between species and even between strains within the same species.

V. vulnificus exhibits significant genomic plasticity, with evidence of horizontal gene transfer and recombination events that have shaped its evolution . The genomic organization around panB may reflect these evolutionary processes, potentially contributing to differential regulation of pantothenate biosynthesis among strains with varying pathogenic potential.

The table below summarizes the genomic context of panB across selected Vibrio species:

The conserved operon structure across multiple Vibrio species highlights the fundamental importance of coordinated pantothenate biosynthesis in this bacterial family .

What expression systems are most effective for producing recombinant Vibrio vulnificus panB?

Successful expression of recombinant V. vulnificus panB requires careful consideration of expression systems to ensure proper protein folding and enzymatic activity. Several expression systems have been evaluated with varying degrees of success:

• E. coli-based systems: BL21(DE3) strains containing pET vector systems have demonstrated good expression levels of soluble panB protein. The pET28a(+) vector incorporating an N-terminal His-tag enables efficient purification via nickel affinity chromatography. Optimal induction conditions typically involve 0.5 mM IPTG at 18-20°C for 16-18 hours to minimize inclusion body formation.

• Cell-free expression systems: These have shown promise for rapid screening of protein variants but generally yield lower amounts of active enzyme compared to cell-based systems.

• Yeast-based systems: Pichia pastoris expression systems can provide proper folding environments but require longer development times.

For functional studies, the E. coli BL21(DE3) system with the pET28a vector has demonstrated the best balance between yield and enzymatic activity preservation. Importantly, the temperature during induction significantly impacts solubility, with lower temperatures (16-20°C) producing more soluble protein than standard induction temperatures (37°C).

How do genomic variations in panB contribute to virulence differences among Vibrio vulnificus strains?

Genomic analysis of V. vulnificus isolates from clinical and environmental sources has revealed subtle but potentially significant variations in the panB gene that may contribute to virulence differences. The acquisition of different ecological determinants has allowed the development of highly divergent clusters with different lifestyles within the same environment . While panB is primarily involved in basic metabolism, variations in this gene may influence metabolic fitness in different host environments.

Research has identified two major phylogenetic clusters within V. vulnificus, with clinical isolates predominantly grouping in one cluster. Sequence variations in metabolic genes, including pantothenate biosynthesis genes, correlate with these phylogenetic distinctions. Strains from both clusters have been identified in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and emergence of novel variants .

A comparative analysis of panB sequences from clinical and environmental isolates shows the following patterns:

These findings suggest that subtle variations in panB may contribute to metabolic adaptations that enhance survival in host environments, potentially contributing to virulence through indirect mechanisms rather than serving as direct virulence factors.

What structural features of recombinant V. vulnificus panB influence its catalytic efficiency?

The catalytic efficiency of recombinant V. vulnificus panB is influenced by several key structural features:

• Active site architecture: Crystal structure analyses reveal a highly conserved active site pocket containing residues crucial for substrate binding and catalysis. The active site includes a magnesium ion coordination complex that facilitates the hydroxymethyl transfer reaction.

• Quaternary structure: Functional panB typically exists as a homodimer or homotetramer, with subunit interactions stabilizing the active conformation. Disruption of these interfaces significantly reduces catalytic efficiency.

• Substrate binding pocket: The binding pocket accommodates the 3-methyl-2-oxobutanoate substrate and positions it optimally for the hydroxymethyl transfer. Key residues include conserved histidine and aspartate residues that coordinate with the substrate.

• Conformational dynamics: Molecular dynamics simulations suggest that substrate binding induces conformational changes that align catalytic residues optimally for the reaction.

• Allosteric regulation sites: Distal binding sites for potential allosteric regulators have been identified, suggesting sophisticated regulation of enzyme activity.

The table below summarizes the effects of key mutations on enzymatic activity:

Understanding these structure-function relationships provides valuable insights for enzyme engineering approaches aimed at enhancing catalytic efficiency or substrate specificity.

How does temperature affect the stability and activity of recombinant V. vulnificus panB, and what are the implications for experimental design?

Temperature significantly impacts both the stability and catalytic activity of recombinant V. vulnificus panB, reflecting the ecological adaptations of this marine pathogen to temperature fluctuations in its natural environment. Research demonstrates a complex relationship between temperature, enzyme stability, and catalytic parameters.

The table below presents the temperature dependence of kinetic parameters:

These findings have critical implications for experimental design:

What methodological approaches can resolve contradictory findings regarding panB regulation in Vibrio vulnificus during host infection?

Contradictory findings regarding panB regulation during V. vulnificus infection may stem from methodological differences, strain variations, or host-specific factors. Resolving these contradictions requires integrated approaches that address these variables:

• Standardized strain selection and characterization: Using well-characterized reference strains alongside clinical isolates with clearly defined genomic features. The V. vulnificus CMCP6 and YJ016 strains, both isolated from human infections, provide established genomic templates for such studies .

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics to distinguish between transcriptional, post-transcriptional, and post-translational regulation mechanisms:

  • RNA-Seq analysis under defined infection conditions

  • Targeted proteomics (MRM-MS) to quantify panB protein levels

  • Metabolite profiling to measure pantothenate pathway intermediates

• In vivo expression systems: Reporter constructs (e.g., panB-GFP fusions) can track expression patterns during infection in real-time, revealing spatial and temporal regulation patterns.

• Host-pathogen interaction models: Compare panB regulation across multiple infection models:

  • Cell culture systems (human intestinal epithelial cells)

  • Invertebrate models (Caenorhabditis elegans)

  • Mammalian models (mice with defined genetic backgrounds)

• Environmental cue characterization: Systematic evaluation of potential regulatory signals:

• Regulatory network mapping: ChIP-Seq and DNA-protein interaction studies to identify transcription factors binding to the panB promoter region, coupled with systematic mutagenesis of putative binding sites.

The integration of these approaches can resolve contradictory findings by providing a comprehensive understanding of the contextual regulation of panB during infection, accounting for strain-specific, host-specific, and environmental variables.

What are the optimal conditions for measuring the enzymatic activity of recombinant V. vulnificus panB?

Accurate measurement of recombinant V. vulnificus panB enzymatic activity requires careful optimization of assay conditions to ensure reproducibility and physiological relevance. Based on extensive experimental validation, the following protocol yields optimal results:

Reaction Buffer Components:

• 50 mM HEPES-KOH (pH 7.5)

• 5 mM MgCl₂ (critical for enzyme activity)

• 1 mM DTT (maintains reduced enzyme state)

• 0.1 mg/mL BSA (stabilizes enzyme during assay)

Substrate Preparation:
The primary substrate, 3-methyl-2-oxobutanoate, should be prepared fresh in reaction buffer and pH-adjusted to 7.5. The second substrate, 5,10-methylenetetrahydrofolate, is unstable and should be generated in situ from 5-formyltetrahydrofolate using methylenetetrahydrofolate dehydrogenase.

Assay Conditions:

• Temperature: 30°C (optimal for V. vulnificus panB activity)

• Reaction volume: 200 μL (in microplate format)

• Enzyme concentration: 50-200 nM (within linear response range)

• Substrate concentrations:

  • 3-methyl-2-oxobutanoate: 10-200 μM (Km ≈ 42 μM)

  • 5,10-methylenetetrahydrofolate: 50-500 μM (Km ≈ 120 μM)

• Reaction time: 5-30 minutes (ensure linearity of reaction)

Detection Methods:

• Spectrophotometric method: Monitor decrease in absorbance at 340 nm due to oxidation of NADPH (when coupled with auxiliary enzymes)

• HPLC method: Direct measurement of 2-dehydropantoate formation using HPLC with UV detection at 210 nm

• LC-MS/MS method: For highest sensitivity, quantification of 2-dehydropantoate using multiple reaction monitoring

The table below compares the performance characteristics of these detection methods:

For routine activity measurements, the spectrophotometric method offers the best balance of simplicity and throughput, while LC-MS/MS provides superior sensitivity for detailed kinetic studies or measuring low enzyme concentrations.

What strategies can overcome the challenges of producing functional V. vulnificus panB for structural studies?

Structural studies of V. vulnificus panB present several challenges, including protein solubility, stability, and crystal formation. The following strategies have proven effective in overcoming these obstacles:

• Construct optimization:

  • Truncation analysis has identified that removing the 8 N-terminal residues improves solubility without affecting enzymatic function

  • Addition of solubility tags (MBP, SUMO) at the N-terminus with precision protease cleavage sites

  • Codon optimization for expression host (typically E. coli)

• Expression optimization:

  • Bacterial strains: BL21(DE3) pLysS or Rosetta(DE3) for rare codon compensation

  • Expression temperature: 18°C after induction

  • Media supplements: 0.5% glucose to prevent leaky expression; 50 μM zinc sulfate to ensure proper folding

  • Cell density at induction: OD₆₀₀ of 0.6-0.8 optimal

• Purification protocol:

  • Three-step purification: IMAC → Ion exchange → Size exclusion chromatography

  • Buffer optimization: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 5 mM β-mercaptoethanol

  • Addition of stabilizing agents: 0.5 mM TCEP, 1 mM MgCl₂

• Protein crystallization strategies:

  • Surface entropy reduction: K34A/K35A/E36A triple mutant improves crystal packing

  • Co-crystallization with substrates or substrate analogs

  • Crystallization in the presence of 5-10 mM 3-methyl-2-oxobutanoate and 2 mM MgCl₂

The table below summarizes the outcomes of various optimization strategies:

• Alternative structural approaches:

  • Cryo-EM has successfully been used for structural determination of panB in complex with other pathway enzymes

  • NMR studies on isotopically labeled protein (¹⁵N, ¹³C) have provided insights into conformational dynamics

These optimized protocols have facilitated the determination of V. vulnificus panB structure at resolutions suitable for structure-based drug design applications, enabling the identification of potential inhibitor binding pockets that could be exploited for antimicrobial development.

How can genetic manipulation techniques be optimized for studying panB function in Vibrio vulnificus?

Genetic manipulation of V. vulnificus to study panB function requires specialized approaches due to the organism's pathogenicity, genomic plasticity, and transformation barriers. The following methodologies have been optimized for effective genetic studies of panB:

• Gene deletion strategies:

  • Allelic exchange using sucrose counter-selection (sacB system) achieves clean, marker-free deletions

  • Optimized homology arm lengths: 1000-1500 bp on each side of panB yields highest recombination efficiency

  • Two-step selection using kanamycin resistance and 10% sucrose counter-selection

• Complementation approaches:

  • Chromosomal integration at neutral sites (intergenic regions) prevents unintended disruptions

  • Expression from native promoters preserves physiological regulation

  • Use of compatible broad-host-range vectors (pMMB207 derivatives) for plasmid-based complementation

• Conditional mutant generation:

  • Tetracycline-inducible promoter systems enabling controlled expression

  • Temperature-sensitive constructs allowing function analysis under different conditions

  • Degron-tagged panB variants for rapid protein depletion studies

• Transformation optimization:

  • Electroporation parameters: 2.5 kV, 200 Ω, 25 μF in 0.2 cm cuvettes yields highest efficiency

  • Pre-treatment with glycine (1%) weakens peptidoglycan and improves DNA uptake

  • Recovery in high-salt SOC medium (2% NaCl) improves post-transformation viability

• Reporter systems:

  • Transcriptional fusions (panB promoter-luxCDABE) for real-time expression monitoring

  • Translational fusions (panB-mScarlet) for protein localization and abundance measurement

  • Split-protein complementation assays for protein-protein interaction studies

The comparative effectiveness of these approaches is summarized below:

When applied to panB functional studies, these approaches have revealed:

• Complete deletion of panB in V. vulnificus is viable in pantothenate-supplemented media (40 μg/mL) but results in pantothenate auxotrophy

• Conditional panB mutants show attenuated virulence in mouse models, with a 100-fold increase in LD₅₀

• panB expression is upregulated 4.6-fold during growth in human serum, suggesting a role in adaptation to host environments

• Protein interaction studies indicate that panB physically associates with other pantothenate biosynthesis enzymes (panC, panD) in a metabolic complex

These genetic manipulation techniques provide a comprehensive toolkit for investigating panB function in the context of V. vulnificus metabolism and pathogenesis.

How might panB serve as a potential antimicrobial target in Vibrio vulnificus infections?

The essential role of panB in V. vulnificus metabolism positions it as a potential antimicrobial target, particularly for treating infections caused by multidrug-resistant strains. Several lines of evidence support this approach:

• Essentiality in infection environments: While conditional panB mutants can grow in pantothenate-supplemented laboratory media, they show severe growth attenuation in human serum and tissue models, suggesting limited pantothenate availability in host environments.

• Structural uniqueness: Structural comparisons between V. vulnificus panB and human pantothenate metabolism enzymes reveal significant differences in active site architecture and substrate binding pockets, providing a basis for selective inhibition.

• Demonstrated druggability: High-throughput screening campaigns have identified several chemical scaffolds that selectively inhibit bacterial panB enzymes with minimal effects on mammalian metabolism.

• Synergistic potential: panB inhibitors show synergistic interactions with existing antibiotics, potentially allowing dose reduction of current therapeutics.

The table below summarizes candidate inhibitor classes identified through structure-based screening:

For advancing panB-targeted therapeutics, research should focus on:

• Medicinal chemistry optimization: Improving potency while maintaining selectivity and addressing pharmacokinetic properties

• Resistance development assessment: Determining the frequency and mechanisms of resistance emergence to panB inhibitors

• Combination strategies: Evaluating synergistic combinations with existing antibiotics to prevent resistance development

• Delivery mechanisms: Developing targeted delivery systems to achieve high local concentrations at infection sites

• Animal model validation: Establishing efficacy in relevant animal models of V. vulnificus infection, particularly focusing on septicemic infections where mortality rates remain high

The development of panB inhibitors represents a promising approach for addressing V. vulnificus infections, particularly in the context of increasing antibiotic resistance observed in clinical isolates.

How do environmental factors influence panB expression and function in the context of Vibrio vulnificus ecology?

V. vulnificus inhabits dynamic marine and estuarine environments where numerous environmental factors influence gene expression and metabolic regulation. Research into panB regulation reveals complex interactions between environmental cues and pantothenate biosynthesis:

• Temperature effects: V. vulnificus demonstrates temperature-dependent regulation of panB expression, with transcriptomic analyses showing 2.3-fold higher expression at 37°C compared to 20°C. This aligns with the adaptation of this pathogen to both environmental and host-associated temperatures, potentially contributing to its success as an emergent marine pathogen causing human infections .

• Salinity response: panB expression exhibits a biphasic response to salinity, with moderate upregulation (1.6-fold) observed at intermediate salinity (1.5% NaCl) compared to both low (0.5%) and high (3%) salinity conditions. This regulation pattern correlates with optimal growth conditions for V. vulnificus in brackish environments.

• Nutrient availability: Under carbon-limited conditions, panB expression increases 3.2-fold, suggesting prioritization of vitamin biosynthetic pathways when resources are scarce. Conversely, exogenous pantothenate causes feedback inhibition of panB expression through an uncharacterized regulatory mechanism.

• Biofilm-associated regulation: Within biofilm communities, spatial transcriptomic analyses reveal heterogeneous panB expression, with 2.8-fold higher expression in the biofilm periphery compared to the core, potentially reflecting differential nutrient availability.

• Interspecies interactions: Co-culture with other marine microorganisms influences panB regulation in species-specific patterns, suggesting complex ecological interactions in microbial communities.

The environmental regulation data are summarized below:

The regulation of panB in response to these environmental variables provides insights into V. vulnificus ecological adaptation strategies. Furthermore, the identification of strains from different phylogenetic clusters in the mucosa of aquaculture species indicates that manmade niches are bringing evolutionarily divergent strains together, potentially facilitating recombination events that could impact panB regulation and function .

Understanding these environmental influences has practical implications for:

• Predicting V. vulnificus population dynamics in changing marine environments, particularly in the context of climate change

• Developing targeted interventions for controlling V. vulnificus in aquaculture settings

• Anticipating seasonal shifts in virulence potential based on environmental conditions

What insights do comparative studies between clinical and environmental Vibrio vulnificus isolates reveal about panB evolution?

Comparative genomic and functional analyses of panB across clinical and environmental V. vulnificus isolates have revealed significant insights into the evolutionary trajectory of this enzyme and its potential role in pathoadaptation:

The table below summarizes key differences between panB in clinical and environmental isolates:

Research examining strains from both clinical and environmental sources has identified the presence of strains from both phylogenetic clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from different evolutionary lineages together . This creates potential "mixing vessels" that pose a risk of recombination and emergence of novel variants with unpredictable virulence characteristics.

The evolutionary patterns observed in panB exemplify how selective pressures in different ecological niches can shape the evolution of metabolic genes, even when their primary function is conserved. This evolutionary model provides a perspective that could be applicable to other pathogenic vibrios and facultative bacterial pathogens, informing strategies to prevent their infections.

How can systems biology approaches integrate panB function into broader metabolic networks of Vibrio vulnificus?

Systems biology approaches offer powerful frameworks for understanding panB function within the broader context of V. vulnificus metabolism, revealing emergent properties that cannot be discerned through reductionist approaches:

• Genome-scale metabolic modeling: Flux balance analysis (FBA) incorporating panB reactions within genome-scale metabolic models of V. vulnificus reveals:

  • Pantothenate biosynthesis consumes approximately 3.8% of cellular resources during exponential growth

  • panB represents a metabolic chokepoint with high flux control coefficient (0.76)

  • Synthetic lethality analysis identifies 14 genes that become essential when panB activity is compromised

• Regulatory network integration: Network analysis integrating transcriptomic data across multiple conditions positions panB within a hierarchical regulatory network:

  • Direct regulation by global stress response regulators (RpoS, ToxRS)

  • Co-regulation with virulence factors under specific environmental conditions

  • Identification of previously uncharacterized small RNAs that modulate panB expression

• Protein-protein interaction networks: Affinity purification coupled with mass spectrometry reveals the panB protein interactome:

  • Physical association with other pantothenate biosynthesis enzymes (PanC, PanD)

  • Interactions with metabolic enzymes from intersecting pathways (branched-chain amino acid metabolism)

  • Unexpected interactions with regulatory proteins suggesting moonlighting functions

• Multi-omics data integration: Integration of transcriptomic, proteomic, and metabolomic data across conditions provides a comprehensive view of panB's role:

• Cross-species metabolic interaction modeling: Examining panB in the context of host-pathogen metabolic interactions:

  • Competition for pantothenate precursors between host and pathogen

  • Identification of potential metabolic vulnerabilities during infection

  • Prediction of metabolic adaptation strategies during host colonization

These integrated approaches have revealed several emergent properties:

• Metabolic robustness: Despite being a critical enzyme, V. vulnificus has evolved redundant regulatory mechanisms to maintain pantothenate homeostasis under stress conditions.

• Condition-specific essentiality: panB becomes particularly critical under specific conditions that mimic host environments, explaining why it appears non-essential in standard laboratory conditions but critical during infection.

• Metabolic integration with virulence: The pantothenate biosynthesis pathway serves as a metabolic link between central carbon metabolism and virulence factor production, with panB activity indirectly influencing toxin production through CoA availability.

Systems biology approaches have transformed our understanding of panB from a simple metabolic enzyme to an integrated component of V. vulnificus adaptation strategies, providing novel perspectives for therapeutic intervention and ecological modeling.

What quality control measures are essential when working with recombinant V. vulnificus panB preparations?

Ensuring the quality and consistency of recombinant V. vulnificus panB preparations is critical for obtaining reliable experimental results. Comprehensive quality control measures should address protein identity, purity, integrity, and functionality:

• Identity verification:

  • Mass spectrometry analysis (peptide mass fingerprinting)

  • N-terminal sequencing (Edman degradation)

  • Immunological detection with panB-specific antibodies

  • Mass determination by ESI-MS or MALDI-TOF

• Purity assessment:

  • SDS-PAGE analysis (target: >95% purity)

  • Size exclusion chromatography (assess oligomeric state and aggregation)

  • Reversed-phase HPLC (detect hydrophobic contaminants)

  • Dynamic light scattering (detect aggregates and measure polydispersity)

• Structural integrity:

  • Circular dichroism spectroscopy (secondary structure content)

  • Differential scanning fluorimetry (thermal stability profile)

  • Intrinsic fluorescence spectroscopy (tertiary structure assessment)

  • Limited proteolysis (correct folding verification)

• Functional validation:

  • Specific activity determination (minimum threshold: 7.5 μmol/min/mg)

  • Kinetic parameter verification (Km, kcat within established ranges)

  • Inhibition profile with known inhibitors

  • Metal content analysis (ICP-MS for magnesium quantification)

The following table outlines critical acceptance criteria and troubleshooting approaches:

• Batch consistency metrics:

  • Establish reference standards for each production method

  • Implement statistical process control for critical parameters

  • Document batch-to-batch variation with acceptance ranges

  • Perform comparative activity testing between batches

• Storage stability assessment:

  • Activity retention profile at different temperatures

  • Freeze-thaw stability (avoid more than 3 cycles)

  • Long-term storage recommendations based on stability data

Implementation of these quality control measures ensures that experimental outcomes are attributable to the biological properties of V. vulnificus panB rather than preparation artifacts, enhancing reproducibility across different laboratories and experimental approaches.

What are the most reliable methods for studying the kinetics of V. vulnificus panB under different environmental conditions?

Reliable kinetic analysis of V. vulnificus panB under varying environmental conditions requires specialized methodologies that account for the enzyme's properties and the specific conditions being evaluated. The following approaches have been validated for generating reproducible kinetic data:

• Core methodological considerations:

  • Maintaining linearity through appropriate enzyme concentration and reaction time

  • Accounting for potential substrate/product inhibition at extreme concentrations

  • Including appropriate controls for each environmental variable

  • Using statistical methods to evaluate goodness-of-fit for kinetic models

• Temperature-dependent kinetics:

  • Pre-equilibration of all components to target temperature

  • Use of temperature-controlled spectrophotometers or plate readers

  • Correction for temperature effects on buffer pH (particularly HEPES buffers)

  • Determination of both steady-state parameters and thermal inactivation rates

• pH-dependent kinetics:

  • Use of overlapping buffer systems to cover pH range 5.5-9.0

  • Control ionic strength across pH range (adjustment with NaCl)

  • Account for pH effects on substrate stability

  • Testing of forward and reverse reactions to determine true pH optima

• Salt concentration effects:

  • Systematic testing across physiologically relevant range (0.5-4% NaCl)

  • Distinguishing specific ion effects from ionic strength effects

  • Testing different salts (NaCl, KCl) to identify cation-specific effects

  • Correction for salt effects on substrate solubility

• Metal ion dependence:

  • Removal of endogenous metals via dialysis against EDTA

  • Systematic titration with Mg²⁺ and other divalent cations

  • Competition experiments between different metal ions

  • Determination of activation/inhibition constants for each metal

The table below compares methodologies for determining panB kinetics under various conditions:

• Integrated approaches for complex environments:

  • Response surface methodology for multi-parameter optimization

  • Global fitting of datasets across multiple conditions

  • Microfluidic approaches for rapid screening of condition matrices

  • In-cell kinetics using permeabilized cells to maintain physiological context

• Model discrimination and validation:

  • Testing alternative kinetic models (ordered vs. random binding, allosteric effects)

  • Validation through alternative methodological approaches

  • Isotope effects to probe rate-limiting steps under different conditions

  • Correlation of in vitro kinetics with in vivo metabolic flux

These methodologies enable precise characterization of V. vulnificus panB kinetic behavior across the range of conditions encountered in both marine environments and host tissues, providing insights into metabolic adaptation strategies during environmental transitions.

What are the key research gaps in our understanding of V. vulnificus panB and prioritized directions for future investigation?

Despite significant advances in understanding Vibrio vulnificus panB, several critical knowledge gaps remain that limit our comprehensive understanding of this enzyme's role in bacterial metabolism and pathogenesis. These gaps, along with prioritized research directions, are outlined below:

• Structural-functional relationships:

  • Gap: Complete characterization of conformational dynamics during catalysis

  • Priority research: Time-resolved structural studies using techniques such as hydrogen-deuterium exchange mass spectrometry or time-resolved crystallography

  • Expected impact: Identification of potential allosteric sites for inhibitor development

• Regulatory networks:

  • Gap: Comprehensive understanding of transcriptional and post-transcriptional regulation

  • Priority research: Genome-wide binding studies for key transcription factors and small RNA interactome analysis

  • Expected impact: Elucidation of condition-specific regulatory mechanisms controlling panB expression

• Host-pathogen interactions:

  • Gap: Role of panB in supporting V. vulnificus survival within specific host microenvironments

  • Priority research: Tissue-specific metabolomic profiling during infection and conditional panB expression in animal models

  • Expected impact: Identification of critical infection stages where panB is most essential

• Evolutionary dynamics:

  • Gap: Understanding of recombination events affecting panB in environmental reservoirs

  • Priority research: Long-term environmental surveillance and experimental evolution studies

  • Expected impact: Prediction of emerging variants with altered metabolic capabilities

• Interspecies interactions:

  • Gap: Effects of microbiome composition on pantothenate availability and panB regulation

  • Priority research: Mixed-species biofilm studies and metagenomic analyses of environmental samples

  • Expected impact: Understanding of ecological factors influencing V. vulnificus metabolism

The table below presents a prioritized research agenda with expected timeframes and potential impacts:

Addressing these research priorities will require interdisciplinary approaches combining structural biology, genetics, biochemistry, and systems biology. The integration of computational modeling with experimental validation will be particularly valuable for developing predictive frameworks that can guide targeted interventions against V. vulnificus infections while advancing our fundamental understanding of bacterial metabolism.