Recombinant Escherichia coli O7:K1 Nicotinate phosphoribosyltransferase (pncB)

What is the biochemical function of nicotinate phosphoribosyltransferase (pncB) in E. coli metabolism?

Nicotinate phosphoribosyltransferase (pncB) catalyzes the conversion of nicotinate (Na) to nicotinate mononucleotide, which is the first reaction in the Preiss-Handler pathway for NAD+ biosynthesis. This enzyme plays a crucial role in maintaining NAD+ homeostasis in E. coli, which is essential for numerous cellular processes including redox reactions, energy metabolism, and various enzymatic activities.

The reaction catalyzed by pncB requires 5-phosphoribosyl-1-pyrophosphate (PRPP) as a co-substrate and magnesium ions as a cofactor. The enzyme transfers the phosphoribosyl group from PRPP to nicotinate, forming nicotinate mononucleotide and releasing pyrophosphate. This reaction is part of the nicotinate salvage pathway, allowing E. coli to recycle nicotinate and maintain adequate NAD+ levels under various environmental conditions .

How does bacterial pncB differ structurally and functionally from human NAPRT?

While bacterial pncB and human NAPRT (Nicotinate phosphoribosyltransferase) catalyze the same chemical reaction, they exhibit several important differences:

• Structural differences:

  • Human NAPRT has additional structural elements not present in bacterial pncB

  • The substrate binding pocket shows differences in amino acid composition, affecting substrate specificity and inhibitor sensitivity

  • Bacterial pncB typically has a simpler domain organization compared to the human enzyme

• Functional differences:

  • Bacterial pncB is unable to activate NF-κB, unlike human NAPRT which can bind to Toll-like receptor 4 (TLR4) and trigger inflammatory signaling

  • Human NAPRT shows a complex pattern of regulation by ATP, exhibiting "apparent dual stimulation/inhibition effect at low/high substrates saturation" following a negative cooperativity model

  • Bacterial pncB generally shows different responses to activators and inhibitors compared to the human enzyme

• Regulatory differences:

  • The expression of bacterial pncB is regulated by different transcriptional networks than human NAPRT

  • Bacterial pncB is often regulated in response to environmental stresses, while human NAPRT regulation is integrated with cellular energy status

These differences can be exploited for developing selective inhibitors that target bacterial pncB without affecting human NAPRT, potentially leading to new antimicrobial strategies .

What are the kinetic parameters of recombinant E. coli pncB?

Recombinant E. coli pncB typically exhibits the following kinetic parameters, though values may vary based on experimental conditions:

Similar to human NAPRT, E. coli pncB may exhibit complex kinetic behaviors, including responses to allosteric modulators such as ATP and inorganic phosphate. These kinetic properties are important considerations when designing activity assays and interpreting experimental results .

What expression systems are most effective for producing recombinant E. coli pncB?

Several expression systems have proven effective for producing recombinant E. coli pncB:

• E. coli expression hosts:

  • BL21(DE3) strain is commonly used due to its high expression levels and lack of proteases

  • Rosetta or Rosetta2 strains can improve expression if rare codons are present in the pncB gene

  • Arctic Express strains may improve protein folding when expressed at lower temperatures

• Vector systems:

  • pET vector systems with T7 promoter provide high-level expression

  • pBAD vectors offer more tunable expression through arabinose induction

  • Dual-tagging strategies (e.g., His-tag and MBP-tag) can improve both purification and solubility

• Induction conditions:

  • Lower temperature induction (16-25°C) often improves solubility

  • IPTG concentration of 0.1-0.5 mM typically provides good balance between yield and solubility

  • Extended expression times (overnight) at lower temperatures can increase yield of properly folded protein

• Fusion partners that enhance solubility:

  • MBP (maltose-binding protein) tag significantly enhances solubility

  • SUMO fusion systems improve both expression and solubility

  • Thioredoxin fusion can improve folding and solubility

The choice of expression system should be guided by the specific research needs, such as the required protein yield, purity, and downstream applications .

What purification strategy provides the highest yield and purity of active recombinant pncB?

A multi-step purification strategy typically yields the best results for recombinant pncB:

Critical considerations for maintaining enzyme activity:

• Buffer components:

  • Include 5-10 mM MgCl₂ in all buffers as it's essential for proper folding and activity

  • Add 1-5 mM DTT or 2-mercaptoethanol to prevent oxidation of cysteine residues

  • Include 10% glycerol to enhance stability during storage

• Handling precautions:

  • Maintain samples at 4°C throughout purification

  • Avoid freeze-thaw cycles by preparing small aliquots for storage

  • Consider adding stabilizers such as PRPP or nicotinate during purification

• Activity verification:

  • Check enzyme activity after each purification step

  • Calculate specific activity (units/mg) to monitor purification progress

  • Verify final product by mass spectrometry and N-terminal sequencing

This strategy typically yields >95% pure pncB with specific activity comparable to the native enzyme .

How can I improve the solubility and stability of recombinant E. coli pncB?

Improving solubility and stability of recombinant pncB can be achieved through several strategies:

• Expression optimization:

  • Lower induction temperature (16-20°C) significantly improves solubility

  • Reduce IPTG concentration to 0.1-0.2 mM

  • Use rich media like Terrific Broth supplemented with extra phosphate

  • Co-express with molecular chaperones like GroEL/GroES or DnaK/DnaJ/GrpE

• Buffer optimization:

  • Include 10-20% glycerol to prevent aggregation

  • Add 1-5 mM DTT or TCEP as reducing agents

  • Optimize salt concentration (typically 150-300 mM NaCl)

  • Maintain pH between 7.5-8.0

  • Include stabilizing agents like trehalose (50-100 mM)

• Storage conditions:

  • Store at high protein concentration (>1 mg/mL) to prevent surface denaturation

  • Add BSA (0.1-1 mg/mL) as a stabilizer for dilute solutions

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

  • Avoid repeated freeze-thaw cycles by preparing small aliquots

• Substrate stabilization:

  • Add substrate or substrate analogs at low concentrations

  • Include 5-10 mM MgCl₂ in all buffers

  • Consider adding inorganic phosphate as it acts as an activator

• Protein engineering approaches:

  • Identify and mutate surface exposed hydrophobic residues

  • Remove flexible regions prone to proteolysis

  • Introduce stabilizing disulfide bonds based on structural information

These approaches can be combined and optimized based on specific requirements for downstream applications .

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

Several methods are available for measuring pncB activity, each with specific advantages:

• Spectrophotometric coupled assays:

  • NAD+ cycling assay: Couples formation of nicotinate mononucleotide to NAD+ production, followed by enzymatic cycling to amplify signal

  • Continuous monitoring of pyrophosphate release using auxiliary enzymes (pyrophosphatase and purine nucleoside phosphorylase)

  • Typical sensitivity: 0.1-1 nmol product formed

• HPLC-based methods:

  • Direct quantification of nicotinate mononucleotide formation

  • Separation on reverse-phase or ion-exchange columns

  • UV detection at 260-280 nm

  • Typical sensitivity: 10-100 pmol product formed

• Radiochemical assays:

  • Using [14C]-nicotinate or [3H]-PRPP as substrates

  • Separation of products by thin-layer chromatography or filtration

  • Highest sensitivity: 1-10 pmol product formed

• Mass spectrometry-based assays:

  • LC-MS/MS for direct quantification of reaction products

  • Can simultaneously monitor substrate depletion and product formation

  • Excellent for detecting multiple reaction products and intermediates

  • Typical sensitivity: 1-10 pmol product formed

Each method has specific advantages depending on the research question:

Proper controls are essential, including no-enzyme controls, heat-inactivated enzyme controls, and substrate specificity controls to ensure accurate activity measurements .

What methods are recommended for studying the structural properties of E. coli pncB?

Several complementary techniques are valuable for investigating the structural properties of pncB:

• X-ray crystallography:

  • Provides atomic-level resolution of protein structure

  • Co-crystallization with substrates, products, or inhibitors reveals binding mechanisms

  • Crystallization conditions typically include:

    • 10-20 mg/mL protein concentration

    • PEG 3350 or ammonium sulfate as precipitants

    • pH range 6.5-8.0

    • Presence of Mg²⁺ and/or substrates to stabilize conformation

• Small-angle X-ray scattering (SAXS):

  • Studies protein conformation in solution

  • Reveals conformational changes upon substrate binding

  • Requires lower protein concentrations than crystallography

• Circular dichroism (CD) spectroscopy:

  • Analyzes secondary structure content (α-helices, β-sheets)

  • Monitors thermal stability and unfolding

  • Assesses structural changes upon ligand binding

• Differential scanning fluorimetry (DSF):

  • Measures thermal stability (melting temperature)

  • Screens for stabilizing conditions and ligands

  • High-throughput format for optimization studies

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps regions of structural flexibility

  • Identifies conformational changes upon substrate binding

  • Detects allosteric effects of ligands

• Site-directed mutagenesis combined with activity assays:

  • Probes functional roles of specific residues

  • Identifies catalytic and substrate-binding residues

  • Elucidates structural basis for allostery

How can I study the interactions between E. coli pncB and potential inhibitors or activators?

Several techniques are valuable for characterizing pncB interactions with modulators:

• Binding assays:

  • Surface plasmon resonance (SPR): Measures real-time binding kinetics, determining kon and koff rates

  • Isothermal titration calorimetry (ITC): Provides thermodynamic parameters (ΔH, ΔS, Kd) of binding

  • Microscale thermophoresis (MST): Detects binding through changes in thermophoretic mobility

  • Fluorescence-based assays using intrinsic tryptophan fluorescence or extrinsic fluorescent probes

• Enzyme kinetics approaches:

  • Inhibition kinetics (determining Ki values and inhibition mechanisms)

  • Activation kinetics (determining Ka values and activation mechanisms)

  • Progress curve analysis for slow-binding inhibitors

  • Global fitting of kinetic data to discriminate between mechanistic models

• Structural studies:

  • X-ray crystallography with bound inhibitors/activators

  • NMR for mapping binding sites and detecting conformational changes

  • Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

• Computational methods:

  • Molecular docking to predict binding modes

  • Molecular dynamics simulations to study binding energetics and conformational changes

  • Virtual screening to identify new potential modulators

An integrated workflow for studying pncB interactions typically includes:

• Initial screening using enzyme activity assays to identify potential modulators

• Kinetic characterization to determine mechanism of action

• Direct binding studies to confirm physical interaction and measure affinity

• Structural studies to elucidate binding mode and molecular interactions

• Site-directed mutagenesis to verify key interaction residues

This comprehensive approach provides a complete picture of how modulators interact with pncB and affect its function, which is valuable for understanding regulation and for potential drug development .

How can transcriptomic and proteomic approaches be used to study pncB regulation in E. coli?

Transcriptomic and proteomic approaches offer powerful tools for investigating pncB regulation:

• Transcriptomic methods:

  • RNA-seq analysis can comprehensively map transcriptional changes in response to various conditions, identifying co-regulated genes and regulatory networks

  • qRT-PCR provides targeted analysis of pncB expression with high sensitivity for detecting subtle changes

  • Microarray analysis can be used to compare pncB expression across multiple conditions simultaneously

  • RNA-seq can also identify transcription start sites and potential regulatory RNA elements affecting pncB expression

• Proteomic methods:

  • Mass spectrometry-based proteomics can quantify pncB protein levels and post-translational modifications

  • Two-dimensional gel electrophoresis can identify changes in pncB expression across different conditions

  • Pulse-chase experiments can determine pncB protein stability and turnover rates

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture) enables precise quantification of protein abundance changes

• Integrative approaches:

  • ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) can identify transcription factors binding to the pncB promoter

  • Ribosome profiling can assess translational efficiency of pncB mRNA

  • Protein-protein interaction studies can identify regulatory partners affecting pncB activity

Example experimental design for studying pncB regulation:

These approaches have revealed that pncB expression can be influenced by various factors including phosphate availability, stress conditions, and NAD+ levels in the cell .

What is known about the role of pncB in bacterial virulence and host-pathogen interactions?

The role of pncB in bacterial virulence is an emerging area of research:

While direct evidence linking pncB to virulence is still emerging, the central role of NAD+ metabolism in bacterial adaptation to host environments suggests pncB may be an important factor in host-pathogen interactions. Further research using pncB knockout strains in infection models would help clarify its specific contributions to virulence.

How can E. coli pncB be utilized in biotechnological applications?

E. coli pncB offers several promising biotechnological applications:

• NAD+ and derivative production:

  • Recombinant pncB can be used in enzymatic synthesis of NAD+ and its derivatives

  • Multi-enzyme cascade systems incorporating pncB can produce high-value nicotinamide-containing compounds

  • Enhanced pncB variants can improve yields in these biocatalytic processes

• Biosensor development:

  • pncB activity can be coupled to reporter systems to create biosensors for:

    • Nicotinate detection in biological samples

    • Screening for pncB inhibitors as potential antimicrobials

    • Monitoring NAD+ metabolism in engineered strains

• Metabolic engineering applications:

  • Overexpression or modification of pncB can enhance NAD+ availability in engineered E. coli

  • Improved NAD+ regeneration systems for whole-cell biocatalysis

  • Engineering the Preiss-Handler pathway for production of novel NAD+ analogs

• Pharmaceutical applications:

  • Structure-based drug design targeting pathogen-specific features of pncB

  • Development of selective inhibitors that target bacterial pncB without affecting human NAPRT

  • High-throughput screening platforms using recombinant pncB

• Protein engineering strategies:

  • Directed evolution of pncB for enhanced thermostability

  • Engineering substrate specificity to accept non-natural substrates

  • Creating fusion proteins with complementary enzymes for cascade reactions

Practical considerations for biotechnological applications:

These applications leverage the catalytic properties of pncB while addressing the challenges of maintaining enzyme activity and stability in various biotechnological contexts .

Why might recombinant E. coli pncB show low or inconsistent enzymatic activity?

Several factors can contribute to low or inconsistent pncB activity:

• Protein folding issues:

  • Rapid overexpression leading to inclusion body formation

  • Improper disulfide bond formation

  • Solution: Lower expression temperature (16-20°C), use specialized folding strains, co-express with chaperones

• Cofactor limitations:

  • Insufficient Mg²⁺ concentration in buffers (optimal range: 5-10 mM)

  • Mg²⁺ chelation by components in the assay buffer (e.g., phosphate, EDTA)

  • Solution: Optimize Mg²⁺ concentration, avoid chelating agents

• Substrate quality and stability:

  • PRPP degradation during storage (half-life ~6 hours at room temperature)

  • Nicotinate purity issues

  • Solution: Prepare fresh PRPP solutions, verify substrate purity by HPLC

• Post-translational modifications:

  • Oxidation of critical cysteine residues

  • Proteolytic damage during expression or purification

  • Solution: Include reducing agents (DTT, TCEP), use protease inhibitors

• Assay interference:

  • Components in the expression/purification system interfering with activity assay

  • Product inhibition during prolonged assays

  • Solution: Additional purification steps, optimize assay conditions

Systematic troubleshooting approach:

By systematically addressing these factors, consistent and high enzyme activity can usually be achieved .

How can I design experiments to differentiate between catalytic and regulatory effects when studying pncB mutations?

Designing experiments to distinguish catalytic from regulatory effects requires a multi-faceted approach:

• Kinetic analysis strategies:

  • Compare kcat/Km values: Catalytic mutations typically affect these parameters

  • Analyze V/K profiles: Regulatory mutations often alter curve shapes rather than just scaling

  • Examine response to allosteric modulators: Regulatory mutations may show altered responses to ATP or phosphate

• Structural and biophysical approaches:

  • Thermal shift assays to detect changes in protein stability

  • Circular dichroism to monitor structural integrity

  • Limited proteolysis to identify conformational changes

  • Hydrogen-deuterium exchange to map dynamic regions

• Mutational design considerations:

  • Alanine scanning of suspected catalytic vs. regulatory sites

  • Conservative vs. non-conservative substitutions

  • Double mutant cycle analysis to detect coupled residues

• Experimental design matrix:

• Controls and validation:

  • Include wild-type enzyme in all experiments

  • Use known catalytic and regulatory mutants as references

  • Perform multiple independent protein preparations

  • Validate findings with complementary methods

This systematic approach can reliably differentiate between mutations affecting the catalytic mechanism versus those altering regulatory properties of pncB, providing insights into structure-function relationships and the molecular basis of enzyme regulation .

What are the major challenges in translating in vitro findings about pncB to its in vivo function in E. coli?

Translating in vitro findings to in vivo contexts presents several challenges:

• Physiological concentrations and conditions:

  • In vitro studies often use substrate concentrations far exceeding physiological levels

  • Cellular concentrations of nicotinate (1-10 μM) and PRPP (50-200 μM) fluctuate with metabolic state

  • Solution: Conduct kinetic studies across physiologically relevant concentration ranges

• Macromolecular crowding effects:

  • Cellular environment contains 200-300 mg/mL macromolecules

  • Crowding affects enzyme kinetics, protein-protein interactions, and substrate diffusion

  • Solution: Include crowding agents (PEG, Ficoll) in in vitro assays

• Complex regulatory networks:

  • pncB functions within integrated metabolic and regulatory networks

  • NAD+ biosynthesis involves multiple pathways with compensatory mechanisms

  • Solution: Systems biology approaches integrating multiple pathways

• Compartmentalization and localization:

  • Microenvironments within bacterial cells affect local substrate concentrations

  • Potential protein-protein interactions in vivo not captured in purified systems

  • Solution: Fluorescence microscopy to study localization, pull-down assays for interaction partners

• Translating genetic manipulation results:

  • Knockout/overexpression studies have pleiotropic effects beyond direct pncB functions

  • Compensatory mechanisms can mask phenotypes in vivo

  • Solution: Conditional expression systems, careful phenotypic analysis, multi-omics approaches

• Environmental adaptation:

  • pncB expression and activity respond to changing environmental conditions

  • Growth phase and stress responses affect NAD+ metabolism

  • Solution: Study pncB under various growth conditions and stress scenarios

To bridge in vitro and in vivo findings, integrative approaches combining biochemical characterization with systems biology are most effective. The use of genetic manipulations (CRISPR-based approaches), metabolic flux analysis, and mathematical modeling can help contextualize in vitro findings within the complex cellular environment .

What are the most promising future research directions for E. coli pncB?

Several promising research directions are emerging for E. coli pncB:

• Structural and mechanistic studies:

  • High-resolution crystal structures of pncB with substrates and regulators

  • Time-resolved structural studies to capture catalytic intermediates

  • Quantum mechanics/molecular mechanics (QM/MM) simulations of the reaction mechanism

• Synthetic biology applications:

  • Engineering pncB variants with novel substrate specificities

  • Development of tunable NAD+ biosynthesis systems

  • Integration of pncB into metabolic circuits for bioproduction

• Systems biology approaches:

  • Multi-omics integration to understand pncB's role in metabolic networks

  • Quantitative modeling of NAD+ homeostasis

  • Genome-scale models incorporating detailed NAD+ metabolism

• Pathogen-host interactions:

  • Investigating pncB's role during infection and colonization

  • Exploring the interplay between pncB and virulence expression

  • Developing pncB inhibitors as potential antimicrobials

• Evolutionary and comparative studies:

  • Analyzing pncB diversity across bacterial species

  • Understanding evolutionary pressures on NAD+ biosynthesis pathways

  • Comparative analysis of regulatory mechanisms across species

These directions will provide deeper insights into NAD+ metabolism, potentially leading to applications in biotechnology, medicine, and basic science .

How does current research on E. coli pncB contribute to our broader understanding of bacterial metabolism?

Research on E. coli pncB contributes to several key areas in bacterial metabolism:

• NAD+ homeostasis mechanisms:

  • Elucidates how bacteria maintain NAD+ levels under variable conditions

  • Reveals regulatory connections between energy metabolism and redox balance

  • Provides insights into metabolic adaptation strategies

• Metabolic network integration:

  • Demonstrates how salvage pathways interface with de novo biosynthesis

  • Illuminates connections between NAD+ metabolism and other cellular processes

  • Reveals regulatory mechanisms coordinating multiple metabolic pathways

• Bacterial stress responses:

  • Shows how NAD+ metabolism adjusts during environmental stress

  • Links metabolic adaptation to survival strategies

  • Connects phosphate limitation responses to NAD+ biosynthesis

• Evolution of metabolic pathways:

  • Provides a model for studying the evolution of substrate specificity

  • Illustrates how salvage pathways complement biosynthetic routes

  • Demonstrates conservation of essential metabolic functions across species

• Host-pathogen metabolic interactions:

  • Reveals how bacterial NAD+ metabolism interfaces with host environments

  • Suggests mechanisms by which bacteria compete for metabolic resources during infection

  • Identifies potential metabolic vulnerabilities in pathogens

These contributions extend beyond E. coli to inform our understanding of bacterial metabolism generally, with implications for antimicrobial development, biotechnology applications, and fundamental microbiology .

What implications might pncB research have for developing new antimicrobial strategies?

Research on pncB offers several promising avenues for antimicrobial development:

• Targeting NAD+ biosynthesis pathways:

  • pncB inhibitors could disrupt bacterial NAD+ homeostasis

  • Structural differences between bacterial pncB and human NAPRT enable selective targeting

  • Combined inhibition of multiple NAD+ biosynthesis enzymes could prevent compensatory mechanisms

• Exploiting bacterial-specific regulatory mechanisms:

  • Compounds disrupting pncB regulation could dysregulate NAD+ metabolism

  • Targeting transcriptional regulators of pncB may affect multiple virulence factors

  • Phosphate-dependent regulation of pncB provides a potential vulnerability

• Structure-based drug design opportunities:

  • High-resolution structures enable rational design of pncB inhibitors

  • Transition state analogs could provide potent and selective inhibition

  • Allosteric inhibitors targeting regulatory sites specific to bacterial pncB

• Combination strategy potential:

  • pncB inhibitors could synergize with existing antibiotics

  • Disrupting NAD+ metabolism may sensitize bacteria to oxidative stress-inducing antibiotics

  • Targeting multiple points in NAD+ biosynthesis could overcome resistance mechanisms

• Biofilm disruption strategies:

  • NAD+ metabolism affects biofilm formation in many bacteria

  • pncB inhibitors might disrupt established biofilms or prevent their formation

  • Combination with anti-biofilm agents could enhance efficacy

These approaches could lead to novel antimicrobials with mechanisms distinct from conventional antibiotics, potentially addressing the growing challenge of antimicrobial resistance .