Recombinant Agrobacterium radiobacter Nicotinate phosphoribosyltransferase (pncB)

What is Nicotinate phosphoribosyltransferase (pncB) and what distinguishes bacterial pncB from mammalian NAPRT?

Nicotinate phosphoribosyltransferase (pncB in bacteria, NAPRT in mammals) catalyzes the conversion of nicotinic acid to nicotinate mononucleotide (NaMN) in the NAD salvage pathway. While both enzymes perform similar catalytic functions, they exhibit important differences:

• Bacterial pncB is not recognized by mammalian NAPRT detection assays, as confirmed in clinical studies of sepsis patients

• Mammalian NAPRT functions as a damage-associated molecular pattern (DAMP) that binds to Toll-like receptor 4 (TLR4) and activates inflammatory responses, independent of its enzymatic activity

• Bacterial pncB lacks the immunomodulatory functions observed in mammalian NAPRT

• Structural and sequence differences between bacterial and mammalian enzymes can be exploited for selective targeting in research applications

What expression systems are most effective for producing recombinant proteins in Agrobacterium?

When expressing recombinant proteins in Agrobacterium, promoter selection is critical. Research has evaluated five commonly used inducible promoters in Agrobacterium strains:

For recombinant pncB expression, the tetracycline-regulated promoter (P tet) is recommended due to its excellent stringency, which is crucial when expressing enzymes that may affect bacterial metabolism .

How can I verify successful expression and activity of recombinant pncB?

Verification of recombinant pncB expression and activity should involve multiple approaches:

• Expression confirmation:

  • Western blot analysis using anti-His tag antibodies (if His-tagged)

  • SDS-PAGE with Coomassie staining to visualize protein of expected size

  • Mass spectrometry for protein identification

• Activity assessment:

  • Enzymatic assay measuring conversion of nicotinic acid to NaMN

  • Spectrophotometric monitoring of reaction progress

  • HPLC analysis of reaction products

  • Comparison of enzymatic parameters with published values

• Functional validation:

  • Complementation assays in pncB-deficient bacterial strains

  • Analysis of NAD levels in cells expressing recombinant pncB

The activity of native eNAPRT has been confirmed to be significantly higher than eNAMPT activity in human plasma samples, which provides a useful benchmark for comparative enzymatic studies .

What recombineering systems can be used for genetic manipulation in Agrobacterium?

Recent advances have developed several recombineering systems specifically for Agrobacterium and related species:

These systems enable precise genome modifications with relatively high efficiency, allowing for targeted gene knockout, replacement, or modification strategies for studying or optimizing pncB expression .

What are the optimal conditions for culturing Agrobacterium for recombinant protein production?

Optimizing Agrobacterium culture conditions for recombinant protein production requires careful attention to several parameters:

• Growth medium: Rich media typically yield higher biomass, but defined media may provide more consistent protein expression

• Temperature: Standard growth at 28°C, but lower temperatures (20-25°C) after induction can improve protein folding

• Induction parameters:

  • For P tet promoter: Optimal tetracycline concentration determined empirically (typically 0.1-1 μg/ml)

  • Induction timing: Mid-log phase typically yields better results than early or late growth phases

• Growth duration: Agrobacterium grows more slowly than E. coli, requiring longer cultivation periods (24-48 hours)

• Aeration: Sufficient aeration is critical for high-density cultures and optimal protein expression

• Plasmid stability: The pBBR1 origin of replication has proven stable in Agrobacterium species and provides a high copy number suitable for recombinant protein expression

How can I optimize homologous recombination efficiency for genetic manipulation of pncB in Agrobacterium?

Optimizing homologous recombination in Agrobacterium for pncB modification requires systematic optimization of several parameters:

• Recombinase system selection: Test multiple RecET-like systems to identify the optimal one for your specific Agrobacterium strain (see table in section 1.4)

• Recombinase expression control:

  • The tetracycline-inducible promoter (P tet) provides precise control of recombinase expression

  • Avoid leaky promoters as unregulated recombinase expression can cause genomic instability

• Homology arm design:

  • Length: 80-bp homology arms have proven effective in Agrobacterium

  • Sequence selection: Avoid repetitive or highly structured regions

  • GC content: Optimize for the GC-rich Agrobacterium genome

• DNA delivery:

  • DNA amount: Higher amounts (up to 4 μg) increase recombination events

  • Electroporation parameters: Optimize field strength and time constant

  • Recovery conditions: Optimize media and recovery time

• Selection strategy:

  • Use appropriate antibiotic selection markers (e.g., apramycin resistance)

  • Screen transformants by colony PCR to confirm successful modifications

In published studies, researchers achieved genomic modifications in various Agrobacterium strains with efficiencies ranging from <100 to approximately 400 colonies per μg of DNA, depending on strain and target .

What experimental approaches can be used to study the structure-function relationship of pncB?

Investigating the structure-function relationship of pncB involves multiple experimental approaches:

• Comparative sequence analysis:

  • Alignment of pncB sequences across bacterial species

  • Identification of conserved catalytic residues

  • Mapping of sequence differences between bacterial pncB and mammalian NAPRT

• Site-directed mutagenesis:

  • Using optimized recombineering systems for Agrobacterium

  • Targeting specific amino acids:

    • Catalytic site residues

    • Substrate binding pocket residues

    • Protein-protein interaction interfaces

• Domain swapping experiments:

  • Creating chimeric proteins with domains from different sources

  • Exchanging domains between bacterial pncB variants

  • Creating bacterial-mammalian hybrid enzymes to study functional differences

• Structural biology approaches:

  • X-ray crystallography of purified pncB

  • Cryo-EM analysis for larger complexes

  • Structure prediction using AlphaFold or similar tools

• Functional validation:

  • Enzyme kinetics assays comparing wild-type and mutant variants

  • Thermal stability assessments

  • Substrate specificity studies

These approaches can provide insights into the molecular basis of pncB function and identify regions that could be targeted for enzyme optimization or inhibitor design.

How does bacterial pncB metabolism integrate with host responses during infection?

While information specific to Agrobacterium radiobacter pncB is limited, research on bacterial-host interactions involving NAD metabolism reveals important considerations:

• Distinction from host inflammatory signaling:

  • Mammalian NAPRT acts as a damage-associated molecular pattern (DAMP) binding to TLR4 and activating NF-κB pathway

  • Mammalian NAPRT enhances macrophage differentiation by inducing macrophage colony-stimulating factor

  • Bacterial pncB is not recognized by NAPRT detection assays and likely does not trigger the same inflammatory responses

• NAD metabolism in infection contexts:

  • NAD is essential for bacterial energy metabolism and virulence factor production

  • Host cells may restrict nicotinate availability as a nutritional immunity strategy

  • Bacterial pncB may be upregulated during infection to compensate for nutrient limitation

• Potential as antimicrobial target:

  • Structural differences between bacterial pncB and mammalian NAPRT could be exploited

  • Inhibitors specific to bacterial pncB could disrupt NAD metabolism

  • Combination approaches targeting multiple steps in bacterial NAD biosynthesis

• Experimental approaches to study pncB in infection:

  • Gene knockout studies using recombineering systems

  • Transcriptomics to analyze pncB expression during infection

  • Metabolomics to track NAD metabolism changes

Understanding these interactions could provide insights into bacterial adaptation strategies and potential therapeutic interventions.

What methodologies are most effective for purifying recombinant pncB from Agrobacterium cultures?

Purification of recombinant pncB from Agrobacterium requires specialized approaches:

• Expression optimization:

  • Use the tetracycline-inducible promoter (P tet) for controlled expression

  • Consider lower induction temperatures to improve protein solubility

  • Evaluate different affinity tags (His, GST, MBP) for optimal purification results

• Cell lysis strategies:

  • Agrobacterium has a complex cell wall requiring robust lysis methods

  • Recommended approaches:

    • High-pressure homogenization

    • Enzymatic treatment with lysozyme combined with mechanical disruption

    • Sonication with optimized protocols

• Purification workflow:

  Step	Method	Considerations
  Capture	Affinity chromatography (IMAC for His-tagged pncB)	Buffer optimization to maintain activity
  Intermediate	Ion exchange chromatography	pH selection based on pncB pI
  Polishing	Size exclusion chromatography	Assessment of oligomeric state
  Quality control	SDS-PAGE, Western blot, Activity assay	Minimum 90% purity for enzymatic studies

• Stability considerations:

  • Addition of stabilizing agents (glycerol, reducing agents)

  • Determination of optimal pH and ionic strength

  • Evaluation of storage conditions (temperature, concentration)

• Activity preservation:

  • Testing of different buffer systems for maintaining enzymatic function

  • Addition of co-factors or substrates for stabilization

  • Assessment of freeze-thaw stability

These optimized protocols can yield high-quality recombinant pncB suitable for structural and functional studies.

How can I develop a high-throughput screening system for pncB inhibitors or enhancers?

Developing high-throughput screening systems for pncB modulators involves multiple methodological considerations:

• Assay development:

  • Spectrophotometric assays monitoring absorbance changes

  • Fluorescence-based approaches for increased sensitivity

  • Coupled enzyme assays with fluorogenic or chromogenic readouts

  • Assay miniaturization for 384 or 1536-well formats

• Screening library considerations:

  • Natural product collections (microbial extracts, plant compounds)

  • Focused libraries targeting NAD metabolism enzymes

  • Fragment-based approaches for initial hits

  • In silico pre-screening based on available structural information

• Screening workflow design:

  Stage	Approach	Throughput	Purpose
  Primary	Single concentration (10-20 μM)	High (10,000+ compounds)	Initial hit identification
  Secondary	Dose-response curves	Medium (500-1000 compounds)	Potency assessment
  Tertiary	Orthogonal assays	Low (50-100 compounds)	Confirmation and specificity
  SAR studies	Analog testing	Very low (10-20 series)	Structure optimization

• Counter-screening strategies:

  • Testing against mammalian NAPRT to identify selective compounds

  • Evaluation of general cytotoxicity

  • Exclusion of pan-assay interference compounds (PAINS)

• Hit validation approaches:

  • Enzyme kinetic studies to determine inhibition mechanisms

  • Thermal shift assays to confirm direct binding

  • Surface plasmon resonance for binding affinity determination

  • Crystallographic studies of enzyme-inhibitor complexes

These approaches provide a comprehensive framework for identifying and characterizing compounds that modulate pncB activity for research or therapeutic applications.

What are the key considerations for engineering pncB for enhanced catalytic properties?

Engineering pncB for enhanced catalytic properties requires strategic approaches based on mechanistic understanding:

• Target properties for enhancement:

  • Catalytic efficiency (kcat/Km)

  • Substrate specificity

  • pH or temperature stability

  • Resistance to inhibition

  • Altered co-factor requirements

• Engineering strategies:

  Approach	Methodology	Advantages	Challenges
  Rational design	Site-directed mutagenesis	Target-specific changes	Requires structural knowledge
  Semi-rational	Saturation mutagenesis of hotspots	Explores sequence space around known sites	Medium-sized libraries
  Directed evolution	Random mutagenesis, DNA shuffling	No structural knowledge needed	Large library screening required
  Computational design	In silico modeling and prediction	Reduces experimental burden	Accuracy limitations

• Implementation using Agrobacterium recombineering:

  • Leverage RecET-like systems for precise genomic modifications

  • Use the tetracycline-inducible promoter (P tet) for controlled expression

  • Design appropriate selection strategies for improved variants

• Screening and characterization:

  • Development of high-throughput activity assays

  • Thermal stability assessment

  • Structural characterization of improved variants

  • Comparative kinetic analysis

• Validation in relevant conditions:

  • Performance under physiological conditions

  • Activity in the presence of potential inhibitors

  • Long-term stability assessments

These approaches provide a framework for developing pncB variants with enhanced properties for biotechnological applications or research tools.