Recombinant Burkholderia vietnamiensis Nicotinate phosphoribosyltransferase (pncB)

What is Nicotinate phosphoribosyltransferase (pncB) and what is its function in Burkholderia vietnamiensis?

Nicotinate phosphoribosyltransferase (NAPRT), encoded by the pncB gene, is an enzyme that catalyzes the rate-limiting step in the NAD salvage pathway starting from nicotinic acid. This enzyme converts nicotinic acid (NA) and phosphoribosyl pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) . In Burkholderia species, including B. vietnamiensis (a member of the Burkholderia cepacia complex), this enzyme plays a critical role in NAD biosynthesis through the Preiss-Handler pathway, which is essential for various cellular processes including energy metabolism and redox reactions .

What are the evolutionary pressures shaping pncB in the Burkholderia cepacia complex?

Research indicates that core orthologous genes in the Burkholderia cepacia complex, which would include pncB, are subject to two major evolutionary forces: recombination and positive selection. Approximately 5.8% of core orthologous genes show evidence of strong recombination, while 1.1% demonstrate positive selection . Homologous recombination contributes significant genetic variation across many genes and maintains genetic cohesion within the Bcc. This high level of recombination between Bcc species blurs taxonomic boundaries, making species difficult to distinguish phenotypically and genotypically . These evolutionary dynamics likely influence pncB's structure and function across different Burkholderia strains.

What are the optimal conditions for expressing recombinant B. vietnamiensis pncB in heterologous systems?

For optimal expression of recombinant B. vietnamiensis pncB, researchers typically use E. coli expression systems with the following considerations:

• Expression vector selection: pET-based vectors with strong inducible promoters are recommended

• E. coli strain: BL21(DE3) or Rosetta strains for proteins with rare codons

• Induction conditions: 0.5-1.0 mM IPTG at mid-log phase (OD600 0.6-0.8)

• Temperature: Reduce to 18-25°C during induction to minimize inclusion body formation

• Expression time: 16-18 hours for maximum yield of soluble protein

Based on published methodologies for recombinant expression of NAPRT orthologs, the coding region of the pncB gene should be amplified from B. vietnamiensis genomic DNA and cloned into an appropriate expression vector with affinity tags to facilitate purification .

What purification strategy yields the highest purity and enzymatic activity for recombinant pncB?

A multi-step purification approach typically yields optimal results:

Throughout purification, it's crucial to maintain the enzyme at 4°C and include protease inhibitors to prevent degradation. For long-term storage, adding 10% glycerol and flash-freezing in liquid nitrogen helps preserve enzymatic activity.

How can researchers troubleshoot expression issues with recombinant B. vietnamiensis pncB?

When encountering expression difficulties with recombinant pncB, consider the following troubleshooting approaches:

• Solubility issues:

  • Lower induction temperature to 16°C

  • Decrease IPTG concentration to 0.1-0.3 mM

  • Add solubilizing agents like 0.1% Triton X-100 to lysis buffer

  • Co-express with chaperone proteins (GroEL/GroES)

• Low expression levels:

  • Optimize codon usage for E. coli

  • Try different fusion tags (MBP, SUMO, GST)

  • Test alternative expression hosts

• Protein instability:

  • Include stabilizing additives (glycerol, reducing agents)

  • Co-express with substrates or substrate analogs

  • Design truncated constructs to remove unstable regions

What are the most reliable assays for measuring B. vietnamiensis pncB enzymatic activity?

Several complementary approaches can be used to measure pncB activity:

• Spectrophotometric assays:

  • Monitor formation of NaMN at 260-280 nm

  • Use coupled enzyme systems that generate NADH for detection at 340 nm

• HPLC-based assays:

  • Direct separation and quantification of reaction products

  • Higher specificity than spectrophotometric methods

• Radiometric assays:

  • Using 14C-labeled nicotinic acid as substrate

  • Most sensitive but requires special handling

The choice depends on required sensitivity, available equipment, and experimental goals. When developing an assay, researchers should optimize substrate concentrations, buffer conditions, and enzyme amounts to ensure linearity within the detection range.

How do pH and temperature affect the enzymatic activity of B. vietnamiensis pncB?

Optimal conditions for B. vietnamiensis pncB activity typically include:

For accurate kinetic measurements, maintaining stable pH is critical as the reaction releases pyrophosphate, which can alter buffer pH. Including a robust buffering system and monitoring pH throughout experiments is recommended.

What are the kinetic parameters of B. vietnamiensis pncB and how do they compare to orthologs from other bacterial species?

While specific kinetic parameters for B. vietnamiensis pncB have not been directly reported in the provided search results, typical values for bacterial NAPRT enzymes include:

These parameters can vary based on experimental conditions and may differ between Burkholderia species. Comparative kinetic analysis with orthologs from other bacterial species can provide insights into evolutionary adaptations and potential targets for selective inhibition.

What structural features are critical for the catalytic activity of B. vietnamiensis pncB?

Based on structural studies of NAPRT enzymes, several key features are likely essential for B. vietnamiensis pncB catalytic activity:

• Substrate binding sites:

  • Nicotinic acid binding pocket typically contains conserved aromatic residues

  • PRPP binding site features positively charged residues that interact with phosphate groups

• Catalytic residues:

  • Conserved aspartate or glutamate residues for acid-base catalysis

  • Lysine or arginine residues that stabilize transition states

• Conformational changes:

  • Domain movements that bring substrates into proximity

  • Induced-fit mechanisms that optimize active site geometry

Structural comparisons with characterized NAPRT enzymes can help identify these critical regions in B. vietnamiensis pncB and guide mutagenesis studies to confirm their roles.

How can researchers model the 3D structure of B. vietnamiensis pncB in the absence of crystallographic data?

In the absence of direct crystallographic data, several computational approaches can predict the structure of B. vietnamiensis pncB:

• Homology modeling:

  • Identify structural templates from related organisms with solved structures

  • Build models using software like SWISS-MODEL, Phyre2, or I-TASSER

  • Validate models through energy minimization and Ramachandran plot analysis

• Molecular dynamics simulations:

  • Refine homology models through equilibration in explicit solvent

  • Identify stable conformations and dynamic regions

  • Simulate substrate binding and potential conformational changes

• Integration with experimental data:

  • Use limited proteolysis to identify domain boundaries

  • Validate models with circular dichroism spectroscopy data

  • Apply site-directed mutagenesis to confirm predicted catalytic residues

What potential inhibitor binding sites exist in B. vietnamiensis pncB that could be exploited for drug development?

Several potential inhibitor binding sites in pncB could be targeted for selective inhibition:

• Competitive inhibitor sites:

  • Nicotinic acid binding pocket for substrate analogs

  • PRPP binding site for nucleotide-like inhibitors

• Allosteric sites:

  • Interface regions between domains

  • Regulatory sites that affect conformational changes

• Unique bacterial features:

  • Binding pockets present in bacterial enzymes but absent in human orthologs

  • Species-specific surface features

Research on benzimidazole derivatives has identified compounds that can modulate NAPRT activity, with compound 18 showing noncompetitive inhibition toward NA (Ki = 338 μM) and mixed inhibition toward PRPP (Ki = 134 μM) . These findings provide starting points for developing more potent and selective inhibitors targeting bacterial pncB enzymes.

How does pncB activity contribute to NAD homeostasis in B. vietnamiensis during infection and stress conditions?

During infection and stress conditions, pncB plays several critical roles in maintaining NAD homeostasis:

• Salvage pathway efficiency:

  • Recycles nicotinic acid from degraded NAD

  • Allows efficient NAD replenishment during rapid growth

  • Provides metabolic flexibility during nutrient limitation

• Redox balance:

  • Ensures sufficient NAD+ pools for cellular redox reactions

  • Supports oxidative stress responses through NADPH generation

  • Maintains energy production under stressful conditions

• Metabolic adaptation:

  • Adjusts NAD synthesis rates to match changing cellular demands

  • Integrates with other metabolic pathways during host adaptation

  • Possibly responds to host-derived signals or metabolites

Understanding these roles can provide insights into bacterial adaptation during infection and identify potential intervention points.

Could extracellular B. vietnamiensis pncB function as an immunomodulatory protein similar to other bacterial NAPRTs?

Recent research has shown that extracellular NAPRT can function as a damage-associated molecular pattern (DAMP) that binds to Toll-like receptor 4 (TLR4) and activates inflammatory responses . In human and mouse macrophages, extracellular NAPRT activates the inflammasome and NF-κB pathways, leading to secretion of inflammatory cytokines . These effects are independent of NAD-biosynthetic activity but rely on NAPRT binding to TLR4 .

This raises the intriguing possibility that B. vietnamiensis pncB could similarly act as an immunomodulatory protein if released extracellularly during infection. Such activity could contribute to:

• Inflammatory responses in infected tissues

• Macrophage activation and differentiation

• Potential role in sepsis-like inflammatory conditions

This would represent a moonlighting function distinct from its primary metabolic role, adding complexity to its role in host-pathogen interactions.

How does inhibition of pncB affect B. vietnamiensis virulence and survival in host environments?

Inhibition of pncB likely impacts B. vietnamiensis virulence and survival through several mechanisms:

• Metabolic consequences:

  • Reduced NAD+ availability limiting energy production

  • Impaired redox balance affecting stress responses

  • Metabolic bottlenecks in NAD-dependent pathways

• Virulence factor expression:

  • Altered regulation of NAD-dependent virulence genes

  • Impaired production of toxins or degradative enzymes

  • Reduced biofilm formation capabilities

• Persistence capabilities:

  • Decreased ability to maintain slow growth during antibiotic exposure

  • Reduced capacity to form persister cells

  • Impaired adaptation to changing host environments

Given that genes involved in material transport and metabolism are favored by selection pressure in Burkholderia species , targeting pncB could potentially disrupt critical metabolic functions required for pathogenesis.

How can recombinant B. vietnamiensis pncB be engineered for improved catalytic efficiency or altered substrate specificity?

Several protein engineering approaches can enhance pncB properties:

• Rational design strategies:

  • Site-directed mutagenesis of active site residues

  • Introduction of stabilizing interactions based on structural analysis

  • Engineering of substrate binding pockets for altered specificity

• Directed evolution approaches:

  • Error-prone PCR to generate variant libraries

  • DNA shuffling with orthologous genes from related species

  • Selection systems based on NAD+ auxotrophy complementation

• Computational design:

  • In silico screening of mutations predicted to enhance stability

  • Molecular dynamics simulations to identify rate-limiting steps

  • Quantum mechanics/molecular mechanics calculations of transition states

Success metrics should include improvements in kcat/Km values, thermal stability, and substrate range expansion.

What high-throughput screening methods can identify novel inhibitors of B. vietnamiensis pncB?

Developing effective high-throughput screening (HTS) assays for pncB inhibitors requires:

• Primary screening assays:

  • Fluorescence-based detection of reaction products

  • Coupled enzyme assays with colorimetric readouts

  • Thermal shift assays to identify direct binders

• Assay optimization parameters:

• Counter-screening strategy:

  • Test hits against human NAPRT to identify selective compounds

  • Eliminate compounds affecting assay components rather than target

  • Confirm mechanism of action through kinetic studies

Promising chemical starting points include benzimidazole derivatives, which have shown potential as NAPRT modulators .

How can systems biology approaches integrate pncB function with broader metabolic networks in B. vietnamiensis?

Systems biology approaches can contextualize pncB function within B. vietnamiensis metabolism through:

• Metabolic flux analysis:

  • 13C labeling studies to track carbon flow through NAD pathways

  • Quantification of metabolic rerouting when pncB is inhibited

  • Integration with genome-scale metabolic models

• Multi-omics integration:

  • Correlation of transcriptomics, proteomics, and metabolomics data

  • Identification of regulatory networks controlling pncB expression

  • Mapping adaptation mechanisms following pncB perturbation

• Network analysis:

  • Identification of synthetic lethal interactions with pncB

  • Mapping of metabolic vulnerabilities during host infection

  • Prediction of combination therapy targets

These approaches can reveal how pncB connects to broader metabolic networks and regulatory systems, providing a systems-level understanding of its role in bacterial physiology and pathogenesis.

What are the main challenges in detecting homologous recombination events affecting the pncB gene in Burkholderia species?

Detecting homologous recombination events in pncB requires sophisticated methodological approaches:

• Statistical procedures for recombination detection:

  • GENECONV analysis with gscale parameter of 1 to allow mismatches within recombining fragments

  • Pairwise homoplasy index (PHI) implementation through PhiPack package

  • Maximum χ2 and neighbor similarity score (NSS) methods

• Sequence analysis challenges:

  • High sequence similarity between Burkholderia species complicating alignment

  • Multiple chromosomal replicons requiring comprehensive genome analysis

  • Gene duplication events potentially confounding ortholog identification

• Validation approaches:

  • Phylogenetic incongruence testing between gene and species trees

  • Analysis of sequence composition biases at potential recombination breakpoints

  • Experimental confirmation through transformation studies

The high level of recombination between Bcc species blurs taxonomic boundaries , making comprehensive analysis crucial for understanding pncB evolution.

How can researchers address protein stability issues when working with purified recombinant B. vietnamiensis pncB?

Protein stability challenges with purified pncB can be addressed through:

• Buffer optimization:

• Storage conditions:

  • Flash-freeze in liquid nitrogen and store at -80°C

  • Add substrate analogs or inhibitors as stabilizing agents

  • Avoid repeated freeze-thaw cycles

• Protein engineering solutions:

  • Identify and remove protease-sensitive regions

  • Introduce stabilizing mutations based on homology models

  • Add fusion partners known to enhance stability

• Analytical techniques:

  • Thermal shift assays to identify stabilizing conditions

  • Size exclusion chromatography to monitor aggregation state

  • Dynamic light scattering to assess homogeneity

What controls are essential when studying potential immunomodulatory functions of extracellular B. vietnamiensis pncB?

When investigating potential immunomodulatory functions of extracellular pncB, several controls are critical:

• Protein quality controls:

  • Endotoxin removal and testing (<0.1 EU/mg protein)

  • Size exclusion chromatography to confirm homogeneity

  • Circular dichroism to verify proper folding

• Functional controls:

  • Catalytically inactive mutants to distinguish enzymatic from immunomodulatory effects

  • Heat-denatured protein to confirm structure-dependent effects

  • Competitive inhibition with TLR4 antagonists

• Cell system validation:

  • Comparison of effects on wild-type and TLR4-deficient cells

  • Dose-response studies to establish physiological relevance

  • Multiple cell types to confirm receptor specificity

• Inflammatory readouts:

  • NF-κB activation by monitoring phosphorylation of IKKα/β, p65 subunit, and ERK1/2

  • Transcription and secretion of pro-inflammatory cytokines including IL1B, IL8, TNFA, and CCL3

  • Assessment of inflammasome stabilization through NLRP3 and Caspase-1 expression

These controls ensure that observed immunomodulatory effects are specific to pncB and not artifacts of experimental conditions.

How might genomic and functional analysis of pncB contribute to understanding Burkholderia cepacia complex taxonomy?

The high level of recombination between Bcc species blurs taxonomic boundaries, making species difficult to distinguish phenotypically and genotypically . Analysis of core genes like pncB could contribute to improved Bcc taxonomy through:

• Genomic approaches:

  • Whole-genome sequence comparisons incorporating recombination analysis

  • Development of multi-locus sequence typing schemes including pncB

  • Identification of species-specific signature sequences within pncB

• Functional characterization:

  • Comparative enzymatic analysis across Bcc species

  • Correlation of pncB variants with metabolic phenotypes

  • Species-specific post-translational modifications or regulation

• Evolutionary analysis:

  • Calculation of selection pressures (dN/dS ratios) across Bcc pncB genes

  • Mapping recombination hotspots within the gene

  • Reconstruction of ancestral sequences and evolutionary trajectories

These approaches could help resolve the taxonomic challenges in the Bcc and provide insights into the evolutionary forces shaping this important bacterial complex.

What are the prospects for developing pncB-targeting antimicrobials with activity against Burkholderia species?

The development of pncB-targeting antimicrobials shows promise based on several considerations:

• Target validation evidence:

  • Essential role of NAD in bacterial metabolism

  • Limited redundancy in NAD biosynthetic pathways

  • Evidence that genes involved in material transport and metabolism are favored by selection pressure

• Drug development strategies:

  • Structure-based design of competitive inhibitors

  • Allosteric inhibitors targeting conformational changes

  • Covalent inhibitors targeting conserved active site residues

• Potential compound classes:

  • Benzimidazole derivatives, which have shown activity as NAPRT modulators

  • Substrate analogs targeting the nicotinic acid binding pocket

  • Rationally designed compounds exploiting differences between bacterial and human enzymes

• Challenges to address:

  • Selectivity over human NAPRT

  • Penetration of the Gram-negative cell envelope

  • Potential for resistance development

How might pncB function in non-pathogenic environmental contexts for B. vietnamiensis?

Beyond its role in pathogenesis, pncB likely serves important functions in environmental adaptation:

• Ecological interactions:

  • NAD metabolism supporting competition with other soil microorganisms

  • Potential role in plant-microbe interactions (B. vietnamiensis is known to associate with plant roots)

  • Contribution to biofilm formation in environmental niches

• Environmental stress responses:

  • Adaptation to fluctuating nutrient availability in soil

  • Response to environmental contaminants and xenobiotics

  • Temperature and pH adaptation in diverse habitats

• Metabolic versatility:

  • Support for the extensive metabolic functions and versatility that allow Bcc species to adapt to a wide range of environments

  • Integration with pathways for degradation of environmental compounds

  • Contribution to nitrogen fixation capabilities in some strains

Understanding these environmental functions could provide insights into the evolution of pncB and its adaptation to different lifestyles.