Recombinant Escherichia coli O127:H6 Nicotinate phosphoribosyltransferase (pncB)

What is the biochemical function of Nicotinate phosphoribosyltransferase in E. coli O127:H6?

Nicotinate phosphoribosyltransferase (pncB) catalyzes the conversion of nicotinic acid (NA) to nicotinic acid mononucleotide (NaMN) using phosphoribosyl pyrophosphate (PRPP) as a co-substrate. This reaction is a critical step in the nicotinamide adenine dinucleotide (NAD) salvage pathway in E. coli. The enzyme plays a pivotal role in maintaining NAD homeostasis, which is essential for numerous redox reactions in cellular metabolism.

To experimentally verify this function, researchers typically employ enzyme assays that measure either the consumption of substrates or the formation of products. A common methodological approach involves spectrophotometric assays that monitor the decrease in PRPP concentration or the increase in NaMN levels. These assays can be conducted using purified enzyme preparations in buffered solutions containing appropriate cofactors and controlled temperature and pH conditions.

How does the expression system affect the yield and activity of recombinant pncB?

The choice of expression system significantly impacts both yield and activity of recombinant pncB. Similar to the methodology used for xylose reductase expression in E. coli, researchers often compare different expression vectors, promoters, and host strains to optimize production .

A systematic approach involves testing multiple expression parameters:

When optimizing expression conditions, researchers should consider that excessive overexpression can lead to inclusion body formation, while insufficient expression results in low yields. Monitoring enzyme activity throughout the optimization process is crucial, as conditions that maximize total protein yield may not necessarily maximize active enzyme yield.

What purification strategies are most effective for recombinant E. coli O127:H6 pncB?

Effective purification of recombinant pncB typically employs a multi-step chromatographic approach. Similar to purification strategies used for other E. coli enzymes, researchers can implement the following methodological workflow:

• Affinity chromatography: Using N-terminal or C-terminal histidine tags (6xHis) allows for initial capture on nickel or cobalt resin columns. Elution is typically performed with an imidazole gradient (50-300 mM).

• Ion exchange chromatography: Based on the theoretical isoelectric point of pncB, either anion exchange (Q-Sepharose) or cation exchange (SP-Sepharose) can be employed as a second purification step.

• Size exclusion chromatography: A final polishing step using Superdex 75 or Superdex 200 columns separates the target protein from aggregates and smaller contaminants.

Researchers should monitor purification efficiency at each step through SDS-PAGE and enzyme activity assays. The specific activity (units of enzyme activity per mg of protein) typically increases with each purification step, while total activity decreases due to inevitable losses during purification.

How can one investigate the substrate specificity of E. coli O127:H6 pncB?

Investigating substrate specificity of pncB requires a systematic approach to test various substrate analogs and quantify their kinetic parameters. A comprehensive methodology includes:

• Substrate analog screening: Test structurally related compounds to nicotinic acid, including nicotinamide, isonicotinic acid, and picolinic acid. For each analog, determine:

  • Relative activity compared to the native substrate

  • Michaelis-Menten parameters (Km, Vmax, kcat)

  • Inhibition constants if they act as competitive inhibitors

• Structure-activity relationship analysis: Correlate chemical properties of the analogs (size, hydrophobicity, charge) with their kinetic parameters to identify key substrate recognition determinants.

• Active site mutagenesis: Based on sequence alignments or structural models, introduce mutations in predicted substrate-binding residues and assess their impact on specificity.

Similar to approaches used in studying xylose transporters in E. coli, researchers can employ isothermal titration calorimetry (ITC) to determine binding constants for different substrates directly . This provides thermodynamic data complementary to the kinetic measurements.

What methodologies are most effective for studying the regulation of pncB expression in response to metabolic changes?

Studying pncB regulation requires a combination of genetic, molecular, and biochemical approaches:

• Promoter analysis: Clone the putative pncB promoter region upstream of a reporter gene (such as lacZ or gfp) and measure expression under different growth conditions. This approach reveals condition-specific regulation patterns.

• Transcription factor identification: Similar to studies on xylose regulon in E. coli, employ DNA-protein interaction techniques such as electrophoretic mobility shift assays (EMSA) to identify transcription factors that bind to the pncB promoter region .

• Metabolite influence assessment: Systematically test the effect of NAD pathway metabolites (NAD+, NADH, nicotinic acid, nicotinamide) on pncB expression using quantitative RT-PCR.

• Global regulatory mechanisms: Employ RNA-seq to compare transcriptomes under different conditions, identifying co-regulated genes that may share regulatory mechanisms with pncB.

For example, in E. coli W3110, researchers have demonstrated that CRP (cyclic AMP receptor protein) influences the expression of numerous metabolic genes in response to carbon source availability . A similar approach could reveal whether pncB expression is under CRP control or influenced by other global regulators.

How can protein engineering be applied to enhance the catalytic efficiency of E. coli O127:H6 pncB?

Protein engineering of pncB can follow several methodological approaches:

• Rational design: Based on structural information or homology models, identify catalytic residues and design mutations to enhance substrate binding or catalytic steps. Key targets include:

  • Residues in the substrate binding pocket

  • Residues involved in PRPP binding

  • Catalytic residues directly participating in the reaction mechanism

• Directed evolution: Create libraries of pncB variants through error-prone PCR or DNA shuffling, then screen for enhanced activity. This approach requires:

  • Development of a high-throughput screening assay for pncB activity

  • Optimization of mutation rates to balance library diversity with functional preservation

  • Multiple rounds of selection and amplification

• Semi-rational approaches: Combine structural insights with targeted randomization of specific regions. For example, site-saturation mutagenesis of residues near the active site.

This multi-faceted approach has been successful for engineering other E. coli enzymes, as demonstrated in studies with xylose reductase, where researchers identified mutations that improved NADPH-dependent activity and selectivity toward xylitol production .

What are the most reliable methods for measuring the kinetic parameters of purified recombinant pncB?

Reliable determination of pncB kinetic parameters requires careful experimental design and multiple complementary approaches:

• Spectrophotometric continuous assays: Monitor the formation of NaMN or consumption of PRPP over time at different substrate concentrations. For accurate results:

  • Maintain temperature control (typically 25°C or 37°C)

  • Buffer at optimal pH (usually pH 7.0-8.0)

  • Include appropriate metal cofactors (often Mg2+ or Mn2+)

  • Use substrate concentrations spanning at least 0.2× to 5× the estimated Km

• Stopped-time assays with HPLC quantification: For more precise product quantification, especially when spectrophotometric signals are weak or obscured by interfering compounds.

• Data analysis approaches: Apply both Lineweaver-Burk plots and non-linear regression to determine Km, Vmax, and kcat values. Non-linear regression typically provides more accurate results, particularly when substrate concentrations close to Km are well-represented in the dataset.

The reliability of kinetic measurements can be assessed through:

• Technical replicates (minimum n=3)

• Multiple enzyme preparations

• Comparison of initial rates at different enzyme concentrations (should scale linearly)

• Controls for product inhibition effects

What are the best approaches for solving structural aspects of E. coli O127:H6 pncB?

Determining the structure of pncB requires a multi-technique approach:

• X-ray crystallography workflow:

  • High-purity protein preparation (>95% by SDS-PAGE)

  • Systematic screening of crystallization conditions (temperature, pH, precipitants)

  • Co-crystallization with substrates, products, or substrate analogs

  • Data collection at synchrotron radiation facilities

  • Phase determination through molecular replacement using related structures

  • Model building and refinement

• Cryo-electron microscopy:

  • Sample preparation on grids with optimal ice thickness

  • Data collection using direct electron detectors

  • Single-particle analysis and 3D reconstruction

  • Resolution enhancement through particle classification

• Complementary structural techniques:

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamic regions

  • NMR spectroscopy for specific domain studies or ligand binding investigations

Structural studies should include both apo-enzyme and enzyme-substrate complexes to elucidate the conformational changes associated with catalysis. This approach has been successfully applied to various E. coli enzymes and transporters, revealing important structure-function relationships .

How can metabolic flux analysis be applied to understand the role of pncB in NAD metabolism?

• Isotope labeling experiments:

  • Culture E. coli with 13C-labeled nicotinic acid or glucose

  • Extract and quantify labeled metabolites using LC-MS/MS

  • Calculate flux ratios at metabolic branch points

  • Compare wild-type, pncB overexpression, and pncB knockout strains

• Metabolic modeling approach:

  • Construct a stoichiometric model of E. coli central metabolism including NAD synthesis pathways

  • Constrain the model with experimentally determined uptake and secretion rates

  • Perform flux balance analysis to predict metabolic behaviors

  • Validate predictions with experimental measurements

• Dynamic metabolite profiling:

  • Measure time-course changes in NAD+, NADH, nicotinic acid, and related metabolites

  • Apply systems of ordinary differential equations to model the dynamics

  • Infer rate constants and control coefficients

This metabolic analysis approach is similar to studies conducted with xylose metabolism in E. coli, where researchers examined the contributions of different transport systems and the impact of genetic modifications on metabolic fluxes .

How can CRISPR-Cas9 genome editing be utilized to study pncB function in E. coli O127:H6?

CRISPR-Cas9 genome editing offers precise genetic manipulation capabilities for studying pncB:

• Knockout strategy:

  • Design guide RNAs targeting the pncB coding sequence

  • Provide a repair template with homology arms flanking the target site

  • Screen transformants for successful editing using PCR and sequencing

  • Analyze the phenotypic consequences of pncB deletion on growth rates, NAD levels, and stress responses

• Point mutation introduction:

  • Design guide RNAs targeting specific regions of pncB

  • Provide repair templates containing desired mutations

  • Confirm mutations by sequencing

  • Assess the impact of specific amino acid changes on enzyme function

• Promoter modification:

  • Target the pncB promoter region with guide RNAs

  • Introduce constitutive promoters or regulated promoter systems

  • Measure the effect of altered expression on NAD metabolism

• Multiplex editing:

  • Simultaneously target pncB and related NAD metabolism genes

  • Study epistatic relationships and pathway redundancy

This targeted genetic approach allows researchers to precisely manipulate pncB function while minimizing off-target effects, similar to strategies used for studying other metabolic pathways in E. coli .

What experimental approaches are most effective for studying protein-protein interactions involving pncB?

Multiple complementary techniques can reveal protein-protein interactions involving pncB:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express tagged pncB in E. coli O127:H6

  • Perform gentle lysis to preserve protein complexes

  • Capture pncB and associated proteins using affinity chromatography

  • Identify interaction partners through mass spectrometry

• Bacterial two-hybrid system:

  • Create fusion constructs of pncB with T25 domain of adenylate cyclase

  • Create a library of E. coli proteins fused to T18 domain

  • Screen for protein interactions that reconstitute adenylate cyclase activity

  • Validate positive interactions through targeted experiments

• Biolayer interferometry or surface plasmon resonance:

  • Immobilize purified pncB on sensor chips

  • Flow potential interaction partners over the surface

  • Measure binding kinetics (kon, koff) and affinity (KD)

  • Test the effects of substrates or inhibitors on interaction stability

• In vivo crosslinking:

  • Treat E. coli cells with membrane-permeable crosslinkers

  • Purify pncB complexes under denaturing conditions

  • Identify crosslinked partners through mass spectrometry

  • Confirm interactions through co-immunoprecipitation

By combining multiple approaches, researchers can build confidence in identified interactions and distinguish between stable complexes and transient interactions. This strategy parallels approaches used to study protein interactions in other E. coli metabolic systems .

How can researchers troubleshoot insoluble expression of recombinant E. coli O127:H6 pncB?

Troubleshooting insoluble expression of pncB requires a systematic approach:

• Optimization of expression conditions:

  • Reduce growth temperature (37°C → 30°C → 25°C → 18°C)

  • Decrease inducer concentration

  • Switch to milder promoters with lower expression rates

  • Harvest cells earlier after induction

• Co-expression with molecular chaperones:

  • Test co-expression with GroEL/GroES system

  • Evaluate DnaK/DnaJ/GrpE chaperone system

  • Consider specialized chaperones like trigger factor

• Fusion tag strategies:

  • Test solubility-enhancing fusion partners (MBP, SUMO, Trx)

  • Optimize tag position (N-terminal vs. C-terminal)

  • Include flexible linkers between tag and protein

• Buffer optimization for purification:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test different salt concentrations (100-500 mM NaCl)

  • Include stabilizing additives (glycerol, arginine, trehalose)

The effectiveness of each approach can be quantified by measuring the ratio of soluble to insoluble protein using SDS-PAGE analysis of supernatant and pellet fractions after cell lysis. This methodical troubleshooting approach has been successfully applied to other E. coli recombinant proteins .

What strategies can overcome substrate inhibition or product inhibition during pncB enzymatic assays?

Addressing inhibition issues in pncB enzymatic assays requires both analytical and experimental strategies:

• Identifying inhibition patterns:

  • Perform enzyme kinetics at varying substrate concentrations

  • Construct Lineweaver-Burk and Dixon plots to determine inhibition type

  • Calculate inhibition constants (Ki)

• Assay modification strategies:

  • Use coupled enzyme assays to continuously remove products

  • Implement stopped-time assays with immediate product separation

  • Dilute enzyme sufficiently to work at very low substrate conversion rates

• Enzyme engineering approaches:

  • Identify residues involved in inhibitor binding through structural analysis

  • Introduce mutations that reduce inhibitor affinity while maintaining substrate binding

  • Screen mutant libraries for variants with reduced inhibition

These strategies can be applied to obtain accurate kinetic parameters even in the presence of inhibitory effects, similar to approaches used for other metabolic enzymes in E. coli .

How does pncB from E. coli O127:H6 compare functionally to the enzyme from other E. coli strains and bacterial species?

A comprehensive comparative analysis of pncB across different organisms should include:

• Sequence analysis:

  • Multiple sequence alignment of pncB homologs

  • Phylogenetic tree construction

  • Identification of conserved residues versus strain-specific variations

  • Analysis of selection pressure on different protein regions

• Biochemical comparison:

  • Expression and purification of pncB from multiple strains under identical conditions

  • Side-by-side kinetic characterization (Km, kcat, substrate specificity)

  • pH and temperature optima determination

  • Stability assays (thermal stability, half-life at physiological temperature)

• Structural comparison:

  • Homology modeling if experimental structures are unavailable

  • Comparison of active site architecture

  • Analysis of surface properties and potential interaction interfaces

This comparative approach allows researchers to identify strain-specific adaptations and evolutionarily conserved features, providing insights into the enzyme's fundamental mechanisms and specialized functions in different bacterial contexts. Similar comparative analyses have been performed for other E. coli metabolic enzymes, revealing important functional variations between strains .

How can heterologous expression systems be optimized for large-scale production of active pncB enzyme?

Optimizing heterologous expression systems for pncB production at scale involves:

• Host strain selection:

  • Compare E. coli BL21(DE3), C41(DE3), and Rosetta strains

  • Evaluate eukaryotic expression systems (yeast, insect cells) if bacterial expression is problematic

  • Consider cell-free protein synthesis for difficult-to-express variants

• Bioreactor cultivation strategy:

  • Develop fed-batch protocols to achieve high cell density

  • Implement dissolved oxygen control strategies

  • Optimize feeding rates based on online monitoring of metabolic activity

• Induction protocol optimization:

  • Compare auto-induction media versus controlled inducer addition

  • Test different induction points (OD600 = 0.6, 1.0, 2.0, etc.)

  • Evaluate continuous low-level expression versus pulse induction

• Downstream processing:

  • Develop scalable cell disruption methods

  • Optimize chromatography steps for manufacturing scale

  • Implement quality control metrics for batch consistency

When scaling up production, researchers should monitor both quantity (mg protein per liter culture) and quality (specific activity, purity) at each process development stage. This approach parallels methods used for the production of other recombinant enzymes in E. coli at scale .

How can systems biology approaches reveal the role of pncB in the broader context of E. coli metabolism?

Systems biology provides a framework for understanding pncB in its metabolic context:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and pncB-modified strains

  • Identify regulatory patterns and metabolic adaptations

  • Map changes onto genome-scale metabolic models

• Flux balance analysis:

  • Construct constraint-based models incorporating pncB reaction

  • Perform in silico knockouts and overexpression simulations

  • Predict growth phenotypes under various nutrient conditions

  • Validate model predictions experimentally

• Regulatory network reconstruction:

  • Identify transcription factors affecting pncB expression

  • Map signaling pathways that respond to NAD metabolism perturbations

  • Construct mathematical models of the regulatory circuitry

• Synthetic biology applications:

  • Design genetic circuits incorporating pncB for NAD level control

  • Engineer strains with optimized NAD metabolism for biotechnological applications

  • Create biosensors based on pncB regulation for metabolite detection

This integrative approach provides a comprehensive understanding of how pncB functions within the complex network of cellular metabolism, similar to systems-level analyses conducted for other E. coli metabolic pathways .

What computational approaches can predict substrate specificity and catalytic mechanisms of pncB?

Computational methods offer powerful tools for studying pncB function:

• Molecular dynamics simulations:

  • Construct atomistic models of pncB with substrates bound

  • Simulate protein dynamics over nanosecond to microsecond timescales

  • Identify key substrate-enzyme interactions and conformational changes

  • Calculate binding free energies for different substrates

• Quantum mechanics/molecular mechanics (QM/MM) calculations:

  • Study the electronic structure of the active site during catalysis

  • Calculate reaction energy barriers for proposed mechanisms

  • Compare energetics of alternative catalytic pathways

• Machine learning applications:

  • Develop models to predict substrate specificity from sequence

  • Use neural networks trained on enzymatic reaction databases to suggest novel substrates

  • Apply convolutional networks to identify functional motifs in sequence data

• Docking and virtual screening:

  • Screen compound libraries for potential substrates or inhibitors

  • Rank compounds by predicted binding affinity

  • Identify key pharmacophore features for substrate recognition

These computational approaches complement experimental studies by providing atomistic insights into mechanism and specificity that may be difficult to obtain experimentally. This integrated computational-experimental strategy has been successfully applied to other E. coli enzymes and can reveal fundamental insights into pncB function .