Recombinant Salmonella enteritidis PT4 Nicotinate phosphoribosyltransferase (pncB)

What is Nicotinate phosphoribosyltransferase (pncB) and what is its primary function?

Nicotinate phosphoribosyltransferase (NAPRTase), encoded by the pncB gene, is a key enzyme in the NAD salvage pathway that catalyzes the conversion of nicotinic acid (NA) to nicotinate mononucleotide (NAMN), which serves as a direct precursor of NAD. This reaction represents the rate-limiting step in the NAD salvage pathway . The enzyme plays a crucial role in recycling intracellular NAD breakdown products and utilizing preformed pyridine compounds from the environment to maintain the cell's NAD pool . PncB enables organisms like Escherichia coli to maintain their total NADH/NAD+ intracellular pool by supplementing the de novo pathway with this salvage mechanism .

The enzyme catalyzes the following reaction:
Nicotinic acid + Phosphoribosyl pyrophosphate (PRPP) → Nicotinate mononucleotide (NAMN) + Pyrophosphate

Experimental characterization typically involves HPLC-based assays using reaction conditions including 50 mM Hepes buffer (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2 mM ATP, 5 mM PRPP, and 0.5 mM nicotinic acid . These standardized conditions enable reliable measurement of enzyme activity across various experimental setups.

How does pncB differ from related enzymes in NAD metabolism?

The pncB enzyme (NAPRTase) can be distinguished from other NAD metabolism enzymes by several key characteristics:

These differences are significant for researchers designing specific inhibitors or developing metabolic engineering strategies targeting NAD metabolism, as they provide opportunities for selective intervention in specific pathways.

What are the optimal expression and purification methods for Salmonella enteritidis PT4 pncB?

The successful expression and purification of recombinant pncB require careful optimization of several parameters:

• Expression system: E. coli is the most commonly used heterologous expression system for bacterial pncB proteins. For Salmonella enteritidis PT4 pncB, researchers typically use E. coli BL21(DE3) or similar strains optimized for recombinant protein expression .

• Vector selection: Expression vectors with T7 promoters (like pET28a) containing appropriate affinity tags (typically His₆-tag) facilitate efficient expression and subsequent purification . The pncB gene can be amplified by PCR from genomic DNA and cloned into the NheI and EcoRI sites of pET28a .

• Culture conditions:

  • Growth medium: LB or 2xYT supplemented with appropriate antibiotics

  • Temperature: 30°C for initial growth, often reduced to 18-25°C after induction to enhance soluble protein production

  • Induction: 0.5-1 mM IPTG when culture reaches OD₆₀₀ of 0.6-0.8

  • Post-induction time: 4-16 hours, with overnight expression at lower temperatures often yielding better results

• Purification protocol:

  • Lysis buffer typically contains 50 mM Hepes (pH 7.5), 300 mM NaCl, 10 mM imidazole, 10% glycerol, and protease inhibitors

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the standard first purification step

  • Tag removal can be performed using proteases like TEV if needed for structural studies

  • Further purification by size exclusion chromatography often improves homogeneity

For optimal stability, purified protein should be stored with 5-10% glycerol and reducing agents like 1 mM DTT. Aliquoting and flash-freezing in liquid nitrogen help maintain enzyme activity for extended periods .

What are the most reliable methods for measuring pncB enzymatic activity?

Several established methods can be used to measure pncB (NAPRTase) enzymatic activity with varying degrees of sensitivity and throughput:

• HPLC-based assay (gold standard):

  • This method directly quantifies the NaMN product formed during the reaction

  • Standard reaction mixtures contain 50 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2 mM ATP, 5 mM PRPP, and 0.5 mM nicotinic acid

  • After incubation at 30°C (typically 20 minutes), the reaction products are analyzed on an ion-paired analytical C18 column

  • This approach provides high accuracy and specificity but requires specialized equipment

• Coupled enzyme assays:

  • These assays link pncB activity to the production or consumption of easily detectable compounds like NADH

  • While more convenient for high-throughput screening, they can be subject to interference from compounds affecting the coupling enzymes

• Kinetic parameter determination:

  • For detailed enzymatic characterization, the nicotinic acid concentration is typically varied (0.1-300 μM) while maintaining saturating concentrations of PRPP and ATP

  • When testing substrate specificity for alternate substrates like nicotinamide, higher enzyme concentrations (25 μM) and substrate concentrations (0.03-20 mM) may be necessary due to lower affinity

These methods can be adapted depending on the specific research question, with the HPLC-based assay providing the most reliable direct measurement of enzymatic activity.

How can researchers investigate the impact of pncB on cellular NAD homeostasis?

To investigate the role of pncB in NAD homeostasis, researchers can employ several complementary experimental approaches:

• Genetic manipulation studies:

  • Gene knockout/knockdown: Creating pncB deletion mutants in Salmonella or model organisms

  • Overexpression studies: Expressing pncB under strong promoters or inducible systems

  • Complementation: Reintroducing wild-type or mutant pncB into knockout strains to confirm phenotype specificity

• Metabolic profiling:

  • Measurement of total NAD levels using enzymatic cycling assays or HPLC

  • Determination of NADH/NAD⁺ ratios using fluorometric methods or LC-MS

  • Quantification of related metabolites (NA, NAM, NMN, NaMN) to assess pathway flux

• Growth condition variations:

  • Studies in E. coli have shown that the effects of pncB overexpression vary significantly between growth conditions :

    • In anaerobic chemostat conditions, pncB overexpression increased total NAD levels and decreased the NADH/NAD+ ratio but did not significantly redistribute metabolic fluxes

    • In anaerobic batch (tube) experiments, pncB overexpression led to significant shifts in metabolic patterns, including decreased lactate production and up to 2-fold increases in the ethanol-to-acetate ratio

• Combination with other metabolic interventions:

  • When pncB overexpression is combined with other NADH regeneration strategies (e.g., NAD+-dependent formate dehydrogenase), researchers observed a hybrid metabolic pattern

  • There appears to be a theoretical maximum yield of approximately 4 mol NADH/mol of glucose that cannot be exceeded even with multiple interventions

These approaches provide complementary insights into how pncB contributes to NAD homeostasis under different physiological conditions and genetic backgrounds.

How does the dual function of pncB/NAPRT as a metabolic enzyme and immune modulator work?

Recent research has uncovered a fascinating dual role for NAPRT (encoded by pncB genes in bacteria and their ortholog in humans):

• Traditional metabolic function:

  • Catalyzes the conversion of nicotinic acid to nicotinate mononucleotide in the NAD salvage pathway

  • Essential for maintaining NAD homeostasis in cells

• Novel immunomodulatory function:

  • Extracellular NAPRT can act as a damage-associated molecular pattern (DAMP)

  • Binds to Toll-like receptor 4 (TLR4) on immune cells

  • Activates the inflammasome and NF-κB pathways

  • Stimulates secretion of inflammatory cytokines

  • Enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor

• Mechanism independence:

  • The immunomodulatory effects are independent of NAD-biosynthetic activity

  • Relies specifically on NAPRT binding to TLR4

• Experimental evidence:

  • Treatment of both human and mouse macrophages with recombinant NAPRT activates the NF-κB pathway

  • Effects are observed through phosphorylation of the IKKα/β protein, p65 subunit, and ERK1/2

  • The effects are dose-dependent, with activity observed even at low concentrations (31 ng/ml)

This dual functionality represents a previously unrecognized link between metabolism and immunity, suggesting NAPRT/pncB as a potential target for immunomodulatory therapies or antibacterial strategies.

What is the relationship between pncB overexpression and metabolic flux redistribution?

Research into pncB overexpression has revealed complex effects on cellular metabolism that are highly dependent on growth conditions:

• Anaerobic chemostat conditions:

  • Overexpression of the pncB gene from Salmonella typhimurium in E. coli increased total NAD levels

  • Decreased the NADH/NAD+ ratio

  • Did not significantly redistribute metabolic fluxes

• Anaerobic batch (tube) conditions:

  • The same pncB overexpression led to significant shifts in metabolic patterns

  • Decreased lactate production was observed

  • Increased ethanol-to-acetate (Et/Ac) ratio by up to two-fold

• Combined metabolic engineering approaches:

  • When pncB overexpression was combined with the substitution of native cofactor-independent formate dehydrogenase (FDH) with an NAD+-dependent FDH, researchers observed a hybrid metabolic pattern

  • This combination produced a metabolic profile with high Et/Ac ratio similar to that obtained with the new FDH but with intermediate lactate levels similar to that obtained with pncB overexpression alone

  • Addition of the pncB gene to the FDH system did not further increase the production of reduced metabolites because the system for NADH regeneration had already reached the maximum theoretical yield (approximately 4 mol NADH/mol of glucose)

How do different bacterial species utilize and regulate pncB activity?

Comparative analysis reveals both conservation and diversity in how different bacterial species utilize and regulate pncB:

• Sequence and functional conservation:

  • The core catalytic domain of pncB shows high conservation among related bacterial species, particularly within Enterobacteriaceae

  • Key catalytic residues involved in PRPP binding and nicotinic acid recognition are typically preserved across diverse bacterial lineages

• Species-specific regulatory mechanisms:

  • In some bacterial species, pncB expression is controlled by specialized regulatory proteins such as NrtR

  • The prs-nadV operon arrangement observed in some species suggests coordinated regulation of NAD synthesis and related pathways

  • Regulatory elements like NrtR binding sites are present upstream of some pncB genes, enabling dynamic response to changing metabolic conditions

• Experimental approaches across species:

  • For Acinetobacter baylyi, researchers have cloned pncB into NheI and EcoRI sites of pET28a for expression and characterization

  • For Streptococcus pyogenes, the pncB gene (NAPRT ortholog) has been amplified from genomic DNA for heterologous expression studies

  • Various species show different kinetic parameters and substrate specificities, reflecting adaptation to their specific ecological niches

Understanding these species-specific differences in pncB utilization and regulation is essential for developing targeted antimicrobial strategies or for designing metabolic engineering approaches in different bacterial hosts.

What are the potential therapeutic applications targeting pncB/NAPRT?

Research into pncB/NAPRT has revealed several promising therapeutic applications:

• Antimicrobial strategies:

  • Targeting pncB could disrupt bacterial NAD homeostasis, potentially inhibiting growth of pathogens reliant on the salvage pathway

  • Structural differences between bacterial and human NAPRT enzymes could enable the development of selective inhibitors

  • Combination with inhibitors of other NAD synthesis pathways may prevent metabolic bypassing and enhance efficacy

• Immunomodulatory approaches:

  • Targeting the newly discovered role of NAPRT as a DAMP that interacts with TLR4

  • Blocking NAPRT-TLR4 interactions could reduce harmful inflammation during infection

  • Modulating this pathway might help manage inflammatory conditions without compromising antimicrobial defenses

• Dual-action therapeutics:

  • Compounds that both inhibit bacterial metabolism through pncB and modulate host immunity could address both aspects of infection

  • This approach might be particularly valuable for difficult-to-treat infections where both bacterial growth and excessive inflammation contribute to pathology

• Diagnostic applications:

  • Using pncB activity or expression as biomarkers for specific infections

  • Developing rapid tests based on NAD metabolite profiles

  • Creating biosensors using engineered pncB variants for environmental or clinical monitoring

These therapeutic approaches represent promising alternatives to traditional antibiotic targets, potentially addressing both bacterial viability and immune-mediated pathology during infection.

How can pncB be utilized in metabolic engineering applications?

The pncB enzyme offers several valuable applications in metabolic engineering:

• Enhanced NAD regeneration systems:

  • Overexpression of pncB can increase total NAD levels in bacterial cells

  • This can be particularly valuable in fermentation processes requiring robust redox balance

  • In E. coli, pncB overexpression has been shown to alter the distribution of fermentation products under anaerobic conditions

• Biofuel production optimization:

  • Engineered pncB expression can enhance ethanol production by increasing the ethanol-to-acetate ratio

  • This effect is particularly pronounced in batch fermentation conditions

  • Combinatorial approaches with other NAD-utilizing enzymes can further fine-tune the metabolic flux distribution

• Fine chemical synthesis:

  • Control of NADH/NAD+ ratios through pncB manipulation can direct flux toward specific reduced products

  • This approach enables the production of chiral compounds with high stereoselectivity

  • The effect varies based on cultivation conditions, with batch processes showing more significant metabolic shifts than continuous cultures

• Limitations and considerations:

  • There appears to be a theoretical maximum yield of approximately 4 mol NADH/mol of glucose that cannot be exceeded even with multiple interventions

  • The effect of pncB overexpression varies significantly between steady-state (chemostat) and transient (batch) conditions

  • Combined approaches integrating multiple NAD-related enzymes may require careful balancing to achieve desired metabolic outcomes

These applications highlight the potential of pncB as a valuable tool in the metabolic engineering toolkit, particularly for processes where NAD availability and redox balance are limiting factors.

What emerging technologies are advancing pncB research?

Several cutting-edge technologies are significantly advancing pncB research:

• Advanced structural biology techniques:

  • Cryo-electron microscopy enabling visualization of pncB in different conformational states

  • Time-resolved crystallography capturing enzyme dynamics during catalysis

  • NMR studies revealing ligand-binding dynamics and allosteric mechanisms

• High-throughput screening platforms:

  • Microfluidic systems for miniaturized enzyme assays

  • Droplet-based technologies allowing thousands of reactions to be monitored simultaneously

  • Automated expression and purification workflows increasing throughput for variant analysis

• Computational approaches:

  • Molecular dynamics simulations providing insights into enzyme mechanisms

  • Machine learning algorithms predicting enzyme properties from sequence data

  • Metabolic modeling frameworks integrating pncB into genome-scale networks

• Genome editing tools:

  • CRISPR-Cas systems enabling precise manipulation of pncB in diverse organisms

  • Multiplex genome engineering approaches for creating strain libraries with varying pncB expression levels

  • Site-directed mutagenesis strategies for systematic analysis of structure-function relationships

• Multi-omics integration:

  • Combining transcriptomics, proteomics, and metabolomics data to understand pncB in a systems context

  • Flux analysis techniques quantifying the impact of pncB manipulation on metabolic pathways

  • Single-cell approaches revealing heterogeneity in pncB expression and function

These technological advances are accelerating both fundamental understanding of pncB biology and applied research aimed at leveraging this enzyme for biotechnological and therapeutic applications.

What are the standard protocols for expressing and purifying recombinant pncB?

Standard protocols for expressing and purifying recombinant pncB typically follow these steps:

• Cloning strategy:

  • Amplify the pncB gene by PCR from genomic DNA using appropriate primers

  • Clone into expression vector (e.g., pET28a) using restriction sites such as NheI and EcoRI

  • Verify construct by sequencing before transformation into expression host

• Expression conditions:

  • Transform verified construct into E. coli BL21(DE3) or similar strain

  • Grow transformants in LB medium with appropriate antibiotic at 37°C until OD600 reaches 0.6-0.8

  • Induce protein expression with 0.5 mM IPTG

  • Continue cultivation at 25-30°C for 4-6 hours or at 18°C overnight

  • Harvest cells by centrifugation (5,000 × g, 10 min, 4°C)

• Cell lysis and initial purification:

  • Resuspend cell pellet in lysis buffer (50 mM Hepes pH 7.5, 300 mM NaCl, 10 mM imidazole, 10% glycerol, protease inhibitors)

  • Lyse cells by sonication or French press

  • Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

  • Apply clarified lysate to Ni-NTA affinity column pre-equilibrated with lysis buffer

• Affinity chromatography and further purification:

  • Wash column with wash buffer (lysis buffer with 20-30 mM imidazole)

  • Elute protein with elution buffer (lysis buffer with 250-300 mM imidazole)

  • For higher purity, perform size exclusion chromatography using buffer containing 50 mM Hepes pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Quality control and storage:

  • Assess purity by SDS-PAGE (>85% purity is typically achievable)

  • Confirm identity by Western blotting or mass spectrometry

  • Measure specific activity using standard enzyme assays

  • Store purified protein at -80°C in buffer containing 50% glycerol or flash-freeze aliquots in liquid nitrogen

Following these protocols should yield functional recombinant pncB suitable for biochemical and structural studies.

How can researchers accurately measure the kinetic parameters of pncB?

Accurate measurement of pncB kinetic parameters requires careful experimental design and data analysis:

• Reaction setup for initial rate determination:

  • Prepare reaction mixtures containing:

    • 50 mM Hepes buffer (pH 7.5)

    • 10 mM MgCl₂

    • 1 mM DTT

    • 2 mM ATP

    • 5 mM PRPP (saturating concentration)

    • Variable concentrations of nicotinic acid (typically 0.1-300 μM)

    • Purified enzyme at appropriate concentration (typically 1-10 nM)

  • Incubate at optimal temperature (usually 30°C)

  • Ensure measurements are made within the linear range of product formation

• Product quantification methods:

  • For HPLC-based assays:

    • Quench reactions at appropriate time points

    • Analyze on ion-paired analytical C18 column

    • Quantify NaMN product using appropriate standards

  • For spectrophotometric assays:

    • Use continuous monitoring if employing coupled enzyme systems

    • Ensure coupling enzymes are not rate-limiting

• Data analysis for kinetic parameter determination:

  • Plot initial velocity versus substrate concentration

  • Fit data to appropriate kinetic model:

    • Michaelis-Menten equation for standard kinetics

    • Modified equations for substrate inhibition (as observed for some pncB variants with nicotinamide)

  • Use software like GraphPad Prism for nonlinear regression analysis

• Determination of all relevant parameters:

  • Km: Measure the substrate concentration at half-maximal velocity

  • kcat: Calculate the turnover number from Vmax and enzyme concentration

  • kcat/Km: Calculate the catalytic efficiency

  • Ki: Determine inhibition constants if substrate inhibition is observed

• Validation and controls:

  • Verify linearity with respect to enzyme concentration and time

  • Include appropriate positive and negative controls

  • Test reproducibility across multiple enzyme preparations

Following these methodological guidelines ensures reliable and reproducible determination of kinetic parameters, facilitating meaningful comparisons between different pncB variants or experimental conditions.

What are the most significant unanswered questions in pncB research?

Despite substantial progress in understanding pncB biology, several important questions remain unanswered:

• Structural dynamics during catalysis:

  • How does pncB undergo conformational changes during substrate binding and product release?

  • What are the rate-limiting steps in the catalytic cycle?

  • How do allosteric effectors modify enzyme activity at the molecular level?

• Evolutionary adaptations:

  • Why have different bacterial species evolved variations in pncB structure and regulation?

  • What selective pressures have shaped pncB substrate specificity across different organisms?

  • How did the dual functionality as both metabolic enzyme and immune modulator evolve?

• Systems-level integration:

  • How is pncB activity coordinated with other NAD synthesis pathways in vivo?

  • What mechanisms maintain NAD homeostasis when pncB function is altered?

  • How does pncB expression change in response to environmental stressors?

• Therapeutic applications:

  • Can selective inhibitors of bacterial pncB be developed without affecting human NAPRT?

  • How can the immunomodulatory function of pncB/NAPRT be therapeutically targeted?

  • What combination approaches might overcome metabolic redundancy in NAD synthesis?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, immunology, and systems biology. The answers will not only advance our fundamental understanding of NAD metabolism but also open new avenues for therapeutic intervention and biotechnological applications.

How is the field of pncB research likely to evolve in the coming years?

The field of pncB research is poised for significant advances in several directions:

• Integration with systems biology:

  • More comprehensive modeling of NAD metabolism incorporating pncB in genome-scale metabolic models

  • Multi-omics approaches to understand pncB regulation in different physiological contexts

  • Network analysis revealing non-obvious connections between pncB and other cellular processes

• Expansion of dual-function understanding:

  • Deeper characterization of the mechanism by which pncB/NAPRT activates TLR4

  • Investigation of whether this dual functionality exists in diverse bacterial species

  • Exploration of therapeutic approaches targeting this immune signaling function

• Synthetic biology applications:

  • Designer pncB variants with altered substrate specificity or regulatory properties

  • Integration of engineered pncB into synthetic metabolic pathways

  • Development of pncB-based biosensors for detecting metabolites or environmental conditions

• Translational research advances:

  • Clinical development of pncB inhibitors as novel antimicrobials

  • Immunomodulatory therapeutics targeting the NAPRT-TLR4 interaction

  • Diagnostic tools based on pncB activity or NAD metabolite profiles

• Methodological innovations:

  • High-throughput approaches for characterizing pncB variants

  • In situ studies of pncB activity in living cells

  • Advanced computational models predicting pncB function from sequence

These developments will likely transform pncB from a relatively specialized research area to a more broadly recognized target for both basic science investigations and applied biotechnological and medical applications.