Recombinant Pseudomonas aeruginosa Nicotinate-nucleotide pyrophosphorylase [carboxylating] (nadC)

What is the function of nadC in Pseudomonas aeruginosa?

The nadC gene in Pseudomonas aeruginosa encodes the enzyme nicotinate-nucleotide diphosphorylase (carboxylating) (EC 2.4.2.19), also known as quinolinate phosphoribosyltransferase (QAPRTase). This enzyme is critical in the NAD+ biosynthetic pathway, catalyzing the transfer of a phosphoribosyl group from 5-phosphoribosyl-1-pyrophosphate (PRPP) to quinolinic acid. This reaction is a key step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+), an essential cofactor involved in numerous metabolic processes in P. aeruginosa .

The enzyme plays a crucial role in maintaining the bacterial NAD+ pool, which is essential for energy metabolism, redox reactions, and various cellular processes. In P. aeruginosa, this pathway is particularly important due to the bacterium's versatile metabolism and adaptability to different environments, including host tissues during infection .

What is the enzymatic reaction catalyzed by Nicotinate-nucleotide pyrophosphorylase in P. aeruginosa?

The nicotinate-nucleotide diphosphorylase (carboxylating) in P. aeruginosa catalyzes the following chemical reaction:

nicotinate D-ribonucleotide + diphosphate + CO₂ → pyridine-2,3-dicarboxylate + 5-phospho-alpha-D-ribose 1-diphosphate

This reaction involves three substrates (nicotinate D-ribonucleotide, diphosphate, and CO₂) and yields two products (pyridine-2,3-dicarboxylate and 5-phospho-alpha-D-ribose 1-diphosphate). The enzyme systematically functions as a nicotinate-nucleotide:diphosphate phospho-alpha-D-ribosyltransferase (carboxylating) .

The reaction mechanism involves:

• Binding of nicotinate D-ribonucleotide to the enzyme active site

• Nucleophilic attack by diphosphate

• Carbon dioxide incorporation

• Formation of the carboxylated product

• Release of 5-phospho-alpha-D-ribose 1-diphosphate

Understanding this reaction is essential for designing enzyme assays and developing potential inhibitors for therapeutic purposes.

How does the nadC gene contribute to P. aeruginosa metabolism?

The nadC gene is integral to P. aeruginosa metabolism through its role in NAD+ biosynthesis. As a participant in nicotinate and nicotinamide metabolism , it connects several metabolic pathways:

P. aeruginosa is known for its metabolic versatility and ability to thrive in diverse environments, including those within human hosts during infection. The NAD+ biosynthetic pathway, involving nadC, is critical for maintaining this metabolic flexibility, particularly in nutrient-limited environments encountered during infection .

What structural information is available for P. aeruginosa Nicotinate-nucleotide pyrophosphorylase?

As of the structural data available, multiple crystal structures have been determined for this class of enzymes. According to the Protein Data Bank, several structures have been solved with PDB accession codes including 1QAP, 1QPN, 1QPO, 1QPQ, 1QPR, 1X1O, 2B7N, 2B7P, and 2B7Q . These structures provide valuable insights into the enzyme's:

• Active site architecture

• Substrate binding pockets

• Conformational changes during catalysis

• Potential sites for inhibitor binding

The structural data reveals that the enzyme belongs to the family of glycosyltransferases, specifically the pentosyltransferases . This structural classification is consistent with its catalytic function in transferring a phosphoribosyl group.

Researchers interested in structure-based drug design or understanding the molecular basis of catalysis can utilize these structures to inform their experimental approaches. The availability of multiple structures, likely representing different states of the enzyme (e.g., with various bound substrates or inhibitors), provides a comprehensive structural framework for further investigations.

What are the best methods for cloning and expressing recombinant P. aeruginosa nadC?

When cloning and expressing recombinant P. aeruginosa nadC, researchers should consider the following methodological approach:

• Gene amplification:

  • Design primers based on the P. aeruginosa PAO1 reference genome

  • Include appropriate restriction sites for subsequent cloning

  • Use high-fidelity polymerase (Q5 or Phusion) to minimize PCR errors

  • Optimize PCR conditions for GC-rich P. aeruginosa genomic DNA

• Vector selection:

  • pET vectors (particularly pET28a) provide good expression levels with N- or C-terminal His-tags

  • Consider using pGEX vectors for GST-fusion proteins if solubility is an issue

  • Codon-optimized vectors may improve expression in E. coli systems

• Expression conditions:

  • BL21(DE3) or Rosetta(DE3) E. coli strains are recommended

  • Initial expression tests should compare temperatures (16°C, 25°C, 37°C)

  • Inducer concentration (IPTG) should be titrated (0.1-1.0 mM)

  • Consider auto-induction media for high-density cultures

• Optimization strategies:

  • If inclusion bodies form, co-express with chaperones (GroEL/ES, DnaK/J)

  • For toxic proteins, use tight expression control systems (pBAD, pLysS)

  • Include protease inhibitors during cell lysis to prevent degradation

This methodological approach ensures maximum yield of functional recombinant protein while minimizing common problems such as inclusion body formation or proteolytic degradation.

How can the enzymatic activity of recombinant Nicotinate-nucleotide pyrophosphorylase be measured?

Measuring the enzymatic activity of recombinant P. aeruginosa nicotinate-nucleotide pyrophosphorylase requires careful assay design:

Spectrophotometric assay:

• Monitor the formation of quinolinic acid at 270-280 nm

• Reaction buffer: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 1 mM DTT

• Substrates: nicotinate D-ribonucleotide (100-500 μM), diphosphate (1-5 mM)

• Incubate at 30°C and measure absorbance changes over 5-10 minutes

• Calculate activity using the molar extinction coefficient

HPLC-based assay:

• Separate reaction products on a C18 reverse-phase column

• Mobile phase: 100 mM potassium phosphate (pH 6.0), 5% acetonitrile

• Monitor product formation at 254 nm

• Quantify using standard curves of authentic standards

Coupled enzyme assay:

• Link product formation to a secondary reaction producing a colorimetric or fluorescent readout

• Use phosphoribosyl pyrophosphate synthetase and a colorimetric phosphate detection method

• Monitor reaction progress in real-time

Data analysis considerations:

• Determine kinetic parameters (Km, Vmax, kcat) using Michaelis-Menten or Lineweaver-Burk plots

• Compare enzyme activity under different pH, temperature, and ionic strength conditions

• Include appropriate controls (heat-inactivated enzyme, reaction without substrate)

These methodological approaches allow for accurate and reproducible measurement of enzyme activity, which is essential for characterizing the recombinant protein and screening potential inhibitors.

What expression systems are most efficient for producing recombinant P. aeruginosa nadC?

Several expression systems have been evaluated for producing recombinant P. aeruginosa nadC, each with distinct advantages:

Methodological considerations for optimizing expression:

• Temperature optimization:

  • Lower temperatures (16-25°C) often improve solubility

  • Extended expression times (16-24 hours) at reduced temperatures enhance yield

• Media formulation:

  • Terrific Broth (TB) or auto-induction media typically outperform LB

  • Supplementation with glucose (0.5%) can reduce basal expression

  • Addition of rare element cofactors may improve enzyme activity

• Induction parameters:

  • IPTG concentration should be optimized (typically 0.1-0.5 mM)

  • OD₆₀₀ at induction significantly impacts yield (optimal range: 0.6-0.8)

• Fusion partners:

  • MBP fusion often increases solubility substantially

  • SUMO tags can improve folding and allow native N-terminus after cleavage

The optimal expression system should be determined empirically through small-scale expression trials before scaling up to larger volumes. For most research applications, the E. coli Rosetta(DE3) system with pET vectors and expression at 18°C provides the best balance of yield and functionality.

What purification strategies are recommended for recombinant P. aeruginosa Nicotinate-nucleotide pyrophosphorylase?

Purification of recombinant P. aeruginosa nicotinate-nucleotide pyrophosphorylase requires a multi-step approach to achieve high purity and retain enzymatic activity:

Recommended purification workflow:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol

  • Imidazole gradient elution (20-250 mM) separates target protein from contaminants

  • Include 1 mM DTT to maintain reduced cysteines

• Intermediate purification:

  • Ion exchange chromatography (IEX) using Q-Sepharose

  • Buffer: 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5% glycerol

  • NaCl gradient elution (50-500 mM) provides further purification

• Polishing step:

  • Size exclusion chromatography (SEC) using Superdex 200

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

  • Separates monomeric protein from aggregates and removes remaining impurities

Methodological considerations:

• Tag removal: If necessary, cleave affinity tags using TEV protease between IMAC and IEX steps

• Stability enhancement: Include 10% glycerol and 1 mM DTT in all buffers to maintain stability

• Sample concentration: Use 30 kDa MWCO concentrators, avoiding concentrations >10 mg/mL to prevent aggregation

• Storage conditions: Flash-freeze aliquots in liquid nitrogen and store at -80°C with 20% glycerol

Quality control metrics:

• Purity assessment: SDS-PAGE should show >95% purity

• Activity verification: Specific activity >5 μmol/min/mg protein

• Oligomeric state: Analytical SEC or native PAGE to confirm correct quaternary structure

• Thermal stability: Differential scanning fluorimetry (Tm >40°C indicates stable protein)

This systematic purification approach typically yields 5-10 mg of highly pure and active enzyme per liter of bacterial culture, suitable for structural studies and detailed enzymatic characterization.

How does nadC expression in P. aeruginosa change during infection in various host environments?

The expression of nadC in P. aeruginosa exhibits dynamic regulation during infection, reflecting the bacterium's adaptation to diverse host environments:

Expression patterns in different infection contexts:

P. aeruginosa employs sophisticated mechanisms to modulate nadC expression in response to host-specific signals. During early infection stages, expression levels often increase to support rapid proliferation and metabolic adaptation. In chronic infections, such as those in cystic fibrosis lungs, sustained nadC upregulation contributes to persistent colonization and resistance to host immune defenses .

Methodological approaches for studying nadC expression:

• Transcriptional analysis:

  • qRT-PCR of infected tissues

  • RNA-seq of bacteria isolated from various infection sites

  • Promoter-reporter fusions (GFP, luciferase) for in vivo monitoring

• Protein-level detection:

  • Western blotting with specific antibodies

  • Targeted proteomics using LC-MS/MS

  • Immunohistochemistry of infected tissues

• Functional assessment:

  • Metabolic profiling of NAD+ levels during infection

  • nadC mutant virulence studies in different infection models

  • In vivo imaging of reporter strains

Understanding these expression patterns provides insights into P. aeruginosa's metabolic adaptations during pathogenesis and identifies potential intervention points for novel therapeutic approaches targeting NAD+ biosynthesis.

What is known about inhibitors of P. aeruginosa Nicotinate-nucleotide pyrophosphorylase and their potential as antimicrobial agents?

Research on inhibitors of P. aeruginosa nicotinate-nucleotide pyrophosphorylase reveals several promising candidates with antimicrobial potential:

Classes of inhibitors and their mechanisms:

• Substrate analogs:

  • Quinolinic acid derivatives with modified carboxyl groups

  • PRPP analogs with non-hydrolyzable phosphate bonds

  • Mechanism: Competitive inhibition at substrate binding sites

• Transition state mimics:

  • Compounds resembling the pentacoordinate phosphorus intermediate

  • Typically contain phosphonamidates or phosphonates

  • Mechanism: High-affinity binding to enzyme active site

• Allosteric inhibitors:

  • Small molecules binding to regulatory sites away from the active center

  • Often identified through high-throughput screening approaches

  • Mechanism: Disruption of enzyme conformational dynamics

Structure-activity relationship (SAR) data:

Methodological approaches for inhibitor development:

• Rational design based on crystal structures:

  • Virtual screening against available enzyme structures

  • Structure-based optimization of hit compounds

  • Fragment-based approaches targeting specific binding pockets

• High-throughput screening:

  • Development of colorimetric or fluorescent activity assays

  • Screening of diverse chemical libraries

  • Confirmation of hits using orthogonal assay methods

• Medicinal chemistry optimization:

  • Improving potency through iterative SAR studies

  • Enhancing bacterial penetration and reducing efflux

  • Optimizing pharmacokinetic properties

The potential of these inhibitors as antimicrobial agents depends on their ability to penetrate P. aeruginosa's notoriously impermeable outer membrane and avoid efflux pump-mediated export. Combination approaches with membrane permeabilizers or efflux inhibitors show promise for enhancing the efficacy of nadC inhibitors against this challenging pathogen.

How does the structure and function of P. aeruginosa nadC compare to homologous enzymes in other pathogens?

Comparative analysis of P. aeruginosa nadC with homologous enzymes in other pathogens reveals important evolutionary relationships and functional diversification:

Structural comparison across pathogens:

Despite sequence variations, the core catalytic machinery remains conserved across bacterial species. The crystal structures available in the PDB (including 1QAP, 1QPN, 1QPO, 1QPQ, 1QPR, 1X1O, 2B7N, 2B7P, and 2B7Q) provide valuable insights into these structural relationships .

Functional differences across species:

• Substrate specificity:

  • P. aeruginosa enzyme shows broader substrate tolerance compared to E. coli homolog

  • M. tuberculosis enzyme has higher affinity for quinolinic acid

  • S. aureus enzyme demonstrates altered kinetics due to hexameric structure

• Regulatory mechanisms:

  • Different allosteric regulation patterns in Gram-positive vs. Gram-negative species

  • Variations in feedback inhibition by NAD+ metabolites

  • Species-specific transcriptional control mechanisms

• Metabolic context:

  • Integration into different metabolic networks across pathogens

  • Variable importance in virulence and stress response

  • Different susceptibility to inhibitors

Methodological approaches for comparative studies:

• Sequence-structure-function analysis:

  • Multiple sequence alignment with ConSurf analysis for conservation mapping

  • Homology modeling for species lacking crystal structures

  • Molecular dynamics simulations to compare dynamic properties

• Biochemical characterization:

  • Side-by-side kinetic analysis under identical conditions

  • Thermal stability comparisons using differential scanning fluorimetry

  • Cross-inhibition studies with species-specific inhibitors

• Functional genomics:

  • Complementation studies across species

  • Transcriptional response analysis to metabolic stresses

  • Comparative phenotyping of knockout mutants

These comparative analyses provide crucial insights for developing narrow-spectrum antimicrobials targeting specific pathogens while minimizing impacts on beneficial microbiota.

What role might nadC play in P. aeruginosa virulence and pathogenesis?

The nadC gene and its encoded enzyme play multifaceted roles in P. aeruginosa virulence and pathogenesis through both direct and indirect mechanisms:

Direct contributions to virulence:

• Metabolic fitness during infection:

  • NAD+ biosynthesis supports energy production required for colonization

  • Enables adaptation to nutrient-limited host environments

  • Sustains metabolic activity under oxidative stress conditions imposed by host immune responses

• Biofilm formation:

  • NAD+-dependent processes are crucial for extracellular polymeric substance production

  • Metabolic shifts during biofilm maturation rely on nadC function

  • Contributes to the characteristic antibiotic tolerance of biofilm communities

• Virulence factor production:

  • Many virulence factors require NAD+-dependent steps in their biosynthesis

  • Quorum sensing systems that regulate virulence are linked to cellular NAD+ status

  • Toxin production pathways have heightened energy requirements supported by nadC activity

Evidence from infection models:

P. aeruginosa employs numerous virulence factors during infection, including exotoxins, proteases, and secondary metabolites . The biosynthesis and regulation of many of these virulence determinants are directly or indirectly linked to NAD+-dependent processes, highlighting the central role of nadC in pathogenesis.

Methodological approaches for studying nadC in virulence:

• Genetic manipulation:

  • Construction of nadC deletion, knockdown, and overexpression strains

  • Complementation studies to confirm phenotype specificity

  • Conditional expression systems for temporal control

• Virulence factor analysis:

  • Quantitative assays for pyocyanin, elastase, and rhamnolipid production

  • Biofilm formation assessment using crystal violet staining and confocal microscopy

  • Transcriptional profiling of virulence genes in nadC mutants

• In vivo infection studies:

  • Animal models of acute and chronic infection

  • Competition assays between wildtype and nadC mutants

  • In vivo imaging to track infection progression

Understanding the role of nadC in virulence provides potential targets for anti-virulence strategies that could complement traditional antibiotic approaches against this challenging pathogen.

What are common challenges in expressing and purifying active recombinant P. aeruginosa nadC?

Researchers working with recombinant P. aeruginosa nadC often encounter several challenges that require systematic troubleshooting approaches:

Challenge 1: Poor expression levels

Challenge 2: Inclusion body formation

Challenge 3: Loss of enzymatic activity

Methodological approach to systematic troubleshooting:

• Expression optimization:

  • Design a factorial experiment testing multiple variables (temperature, IPTG concentration, media composition)

  • Use small-scale cultures (10-50 mL) for rapid screening

  • Analyze total and soluble fractions by SDS-PAGE and Western blotting

• Solubility enhancement:

  • If inclusion bodies persist, develop a refolding protocol using gradual dialysis

  • Test additives that promote folding (arginine, sucrose, low concentrations of guanidine)

  • Consider on-column refolding during affinity purification

• Activity preservation:

  • Identify stabilizing buffer conditions through thermal shift assays

  • Minimize freeze-thaw cycles by preparing single-use aliquots

  • Test activity immediately after purification and after storage to establish stability profile

These systematic approaches address the major challenges in working with recombinant P. aeruginosa nadC, enabling researchers to obtain adequate quantities of active enzyme for downstream applications.

What control experiments are essential when studying the effects of nadC mutations on P. aeruginosa physiology?

Designing rigorous control experiments is critical for accurately interpreting the effects of nadC mutations on P. aeruginosa physiology:

Essential control experiments:

• Genetic complementation controls:

  • Wild-type strain (baseline physiology)

  • nadC deletion mutant (knockout phenotype)

  • Complemented strain (nadC mutant with wild-type gene on plasmid)

  • Vector-only control (to account for plasmid burden effects)

  • Site-directed mutants (catalytically inactive but structurally intact)

• Metabolic controls:

  • NAD+ supplementation experiments (bypassing biosynthetic defect)

  • Nicotinamide riboside supplementation (alternate salvage pathway)

  • Measurement of intracellular NAD+/NADH levels (direct metabolic impact)

  • Analysis of upstream and downstream metabolites (pathway flux)

• Phenotypic validation controls:

  • Growth in multiple media conditions (minimal vs. rich)

  • Stress response evaluation (oxidative, nitrosative, antibiotic)

  • Multiple virulence factor assays (to distinguish specific from general effects)

  • Temporal analysis (early vs. late growth phase effects)

Control experiment design matrix:

Methodological considerations for control experiments:

• Strain construction and validation:

  • Confirm genetic manipulations by PCR and sequencing

  • Verify protein expression levels by Western blotting

  • Ensure no unintended mutations through whole-genome sequencing

• Experimental conditions:

  • Standardize growth conditions precisely (temperature, aeration, inoculum)

  • Account for growth rate differences when comparing phenotypes

  • Consider both batch culture and continuous culture approaches

• Statistical analysis:

  • Calculate appropriate sample sizes for adequate statistical power

  • Use repeated measures designs when appropriate

  • Apply correction for multiple comparisons (e.g., Bonferroni, FDR)

Implementation of these control experiments will significantly enhance the reliability and interpretability of studies examining nadC mutations in P. aeruginosa, allowing researchers to distinguish direct enzymatic effects from secondary metabolic or regulatory consequences.

What are the future directions for research on P. aeruginosa nadC?

The study of P. aeruginosa nicotinate-nucleotide pyrophosphorylase (nadC) continues to evolve, with several promising research directions emerging from current understanding:

• Structure-based drug design:

  • Leveraging the multiple crystal structures available (PDB: 1QAP, 1QPN, 1QPO, 1QPQ, 1QPR, 1X1O, 2B7N, 2B7P, 2B7Q)

  • Development of selective inhibitors with reduced host toxicity

  • Rational design of molecules targeting P. aeruginosa-specific structural features

• Systems biology integration:

  • Mapping the complete NAD+ metabolic network in P. aeruginosa

  • Understanding compensatory pathways activated upon nadC inhibition

  • Modeling metabolic flux under different infection conditions

• Host-pathogen interaction studies:

  • Investigating how host NAD+ metabolism interfaces with bacterial requirements

  • Examining nadC expression in polymicrobial infection contexts

  • Studying the role of nadC in adaptation to host immune responses

• Clinical and translational applications:

  • Exploring nadC as a biomarker for metabolic adaptation during chronic infection

  • Developing combination therapies targeting NAD+ biosynthesis and utilization

  • Investigating nadC-targeted therapeutics for multidrug-resistant strains