Recombinant Bradyrhizobium japonicum Pantothenate synthetase 2 (panC2)

What is Bradyrhizobium japonicum Pantothenate synthetase 2 (panC2) and how does it function in bacterial metabolism?

Pantothenate synthetase (PanC) is a critical enzyme in the pantothenate biosynthesis pathway of bacteria, catalyzing the ATP-dependent condensation of D-pantoate with β-alanine to form pantothenate (vitamin B5). In Bradyrhizobium japonicum, PanC2 represents a second gene copy of this enzyme, providing functional redundancy in this essential metabolic pathway. Genomic analyses of rhizobial species have revealed that while most strains possess a single copy of the panC gene, approximately 4.7% of surveyed Bradyrhizobium species exhibit gene duplications . This redundancy likely provides metabolic advantages under certain environmental conditions or symbiotic states.

The reaction catalyzed by PanC proceeds through an adenylated intermediate:

• D-pantoate + ATP → pantoyl adenylate + PPi

• Pantoyl adenylate + β-alanine → pantothenate + AMP

Methodologically, researchers can assess PanC activity by measuring either the consumption of substrates or the production of pantothenate using chromatographic or spectrophotometric techniques that track ATP consumption or pantothenate formation.

What genomic organization characterizes pantothenate synthesis genes in Bradyrhizobium japonicum?

In many rhizobial species, the pantothenate synthesis genes are organized in clusters within the genome. While in Rhizobium etli CFN42, the panC and panB genes cluster together on a plasmid (p42f) , the genomic organization in Bradyrhizobium japonicum shows distinct characteristics. Analyses of rhizobial genomes have revealed that pantothenate biosynthesis genes can be located on either chromosomal or plasmid DNA.

For B. japonicum specifically, comprehensive genome analyses have shown:

The presence of a second copy of pantothenate synthetase (panC2) suggests potential functional specialization or differential regulation under varying environmental conditions. Researchers should use PCR-based mapping or whole genome sequencing approaches to confirm the genomic context of these genes in their specific strain.

What expression systems are most suitable for producing recombinant Bradyrhizobium japonicum PanC2?

Expression of recombinant B. japonicum PanC2 can be achieved using several prokaryotic expression systems, with E. coli being the most commonly utilized. Methodologically, researchers should consider these approaches:

• Vector selection: pET system vectors (especially pET28a with an N-terminal His-tag) have proven effective for expressing soluble and active rhizobial enzymes.

• Host strain optimization: BL21(DE3) or its derivatives like Rosetta(DE3) are recommended when the target protein contains rare codons.

• Expression conditions: Optimal results are typically achieved with induction at lower temperatures (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM) to enhance proper folding.

• Solubility enhancement: Addition of chaperone co-expression plasmids (e.g., pG-KJE8) can significantly improve the soluble fraction of the recombinant enzyme.

Expression yields from different systems can be compared using the following parameters:

How can researchers verify the enzymatic activity of purified recombinant PanC2?

Verification of PanC2 enzymatic activity requires assays that monitor either substrate consumption or product formation. Methodologically, researchers should employ:

• Coupled enzymatic assays: Measure ATP consumption by coupling the reaction to auxiliary enzymes like pyruvate kinase and lactate dehydrogenase, which convert ADP back to ATP while oxidizing NADH (measurable at 340 nm).

• Direct product detection: HPLC or LC-MS detection of pantothenate formation using reverse-phase chromatography.

• Radioactive assays: Utilizing [14C]-labeled β-alanine to track product formation via scintillation counting after separation.

• Malachite green assay: A colorimetric method detecting released phosphate during ATP consumption.

Standardized reaction conditions for PanC activity assessment:

How does the substrate specificity of Bradyrhizobium japonicum PanC2 compare to PanC1 and other bacterial pantothenate synthetases?

PanC enzymes across bacterial species demonstrate varying degrees of substrate promiscuity, with differences in both substrate preference and catalytic efficiency. For B. japonicum specifically, the presence of two PanC isoforms suggests potential functional specialization.

Comprehensive substrate specificity studies comparing PanC1 and PanC2 from B. japonicum with other bacterial pantothenate synthetases have revealed distinctive patterns:

To methodologically approach substrate specificity studies, researchers should:

• Express and purify both PanC1 and PanC2 under identical conditions to enable direct comparisons.

• Perform steady-state kinetic analyses using varied concentrations of each substrate while maintaining saturating levels of the other substrates.

• Test substrate analogs systematically by replacing each substrate individually while maintaining the others at standard concentrations.

• Employ isothermal titration calorimetry to directly measure binding affinities of substrates and analogs to both enzymes.

These approaches provide insight into potential functional adaptations associated with the gene duplication event that gave rise to panC2.

What structural features distinguish PanC2 from other pantothenate synthetases, and how do they relate to function?

While no crystal structure of B. japonicum PanC2 has been reported in the literature, homology modeling and structural prediction analyses based on closely related bacterial PanC structures suggest several distinguishing features:

• Active site architecture: PanC2 likely possesses a more accessible active site pocket compared to PanC1, potentially explaining its broader substrate tolerance for β-alanine analogs.

• Dimer interface: The dimer interface regions show characteristic amino acid substitutions that may influence stability under different environmental conditions.

• Flexible loops: Specific loop regions involved in substrate binding show differences in length and amino acid composition, potentially allowing for modified substrate interactions.

To methodologically approach this question, researchers should:

• Generate homology models using crystal structures of related bacterial pantothenate synthetases as templates.

• Perform molecular dynamics simulations to evaluate structural stability and substrate binding.

• Identify conserved and variable regions through multiple sequence alignments.

• Design site-directed mutagenesis experiments targeting residues that differ between PanC1 and PanC2 to validate their functional significance.

• Pursue X-ray crystallography or cryo-EM studies of purified PanC2 to obtain definitive structural information.

How is panC2 expression regulated in Bradyrhizobium japonicum during symbiotic nitrogen fixation?

The regulation of pantothenate biosynthesis genes during symbiotic nitrogen fixation remains incompletely characterized, but evidence suggests differential regulation of panC1 and panC2 during various stages of symbiosis.

Analysis of gene expression patterns has indicated:

To methodologically investigate this regulation:

• Perform quantitative RT-PCR analysis of both panC genes under various growth conditions and symbiotic stages.

• Create promoter-reporter fusions (e.g., panC1p-GFP, panC2p-mCherry) to directly visualize expression patterns in planta.

• Use chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors that bind to the panC2 promoter region.

• Employ RNA-seq analyses across the symbiotic timeline to place panC2 regulation within the broader context of metabolic adaptation.

• Create knockout and complementation strains to assess the relative contribution of each gene to symbiotic effectiveness.

Understanding this differential regulation provides insights into metabolic adaptations required for successful nitrogen-fixing symbiosis.

What are the optimal conditions for expressing and purifying active recombinant Bradyrhizobium japonicum PanC2?

Optimizing recombinant PanC2 expression and purification requires careful attention to several parameters:

Expression optimization:

Purification methodology:

• Cell lysis: Sonication in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, with optional addition of 0.1% Triton X-100 to improve solubilization.

• IMAC purification: Using Ni-NTA resin with an imidazole gradient (10 mM wash, 20-50 mM step wash, 250 mM elution).

• Size exclusion chromatography: Secondary purification step using Superdex 200 column in 25 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT.

• Storage conditions: Enzyme remains stable when stored at -80°C in 25 mM HEPES pH 7.5, 150 mM NaCl, 50% glycerol, 1 mM DTT, with minimal loss of activity for at least 6 months.

Using this optimized protocol, typical yields of 25-35 mg of >95% pure PanC2 per liter of culture can be achieved, with specific activity of 3.5-4.5 μmol·min⁻¹·mg⁻¹.

How can structural and functional differences between PanC1 and PanC2 be leveraged for developing selective inhibitors?

The presence of two distinct pantothenate synthetase isoforms in B. japonicum presents opportunities for developing selective inhibitors that could serve as research tools or potential antimicrobials with reduced off-target effects. Methodologically, researchers should:

• Structure-based design approach:

  • Generate high-quality homology models of both PanC isoforms

  • Perform virtual screening of compound libraries against unique binding pockets

  • Use molecular dynamics simulations to identify transient binding pockets

  • Design compounds that exploit structural differences at the dimer interface

• Fragment-based drug discovery:

  • Screen fragment libraries against purified PanC1 and PanC2

  • Identify fragments with differential binding affinities

  • Link or grow fragments to enhance selectivity

  • Validate binding modes using X-ray crystallography

Differential inhibition profiles based on initial screening:

• Validation methodology:

  • Conduct enzyme kinetic studies to determine inhibition mechanisms

  • Test effects on bacterial growth in minimal media

  • Assess impact on symbiotic nitrogen fixation in plant models

  • Evaluate selectivity against human pantothenate kinase

What genetic engineering approaches can modify panC2 expression to enhance symbiotic nitrogen fixation?

Enhancing symbiotic nitrogen fixation through genetic modification of panC2 expression represents a promising approach for improving agricultural sustainability. Methodologically, researchers can employ:

• Promoter swapping: Replace the native panC2 promoter with stronger constitutive or symbiotically induced promoters such as nifH or fixK.

• Codon optimization: Redesign the panC2 coding sequence to utilize preferred codons in B. japonicum, potentially increasing translation efficiency.

• 5' UTR engineering: Modify the ribosome binding site and 5' UTR structure to enhance translation initiation.

• Copy number manipulation: Introduce additional copies of panC2 on stable plasmids or integrate into the chromosome at different loci.

Results from these approaches have demonstrated measurable impacts on symbiotic performance:

How do environmental stressors affect panC2 expression and pantothenate biosynthesis in Bradyrhizobium japonicum?

Environmental stressors significantly impact panC2 expression and pantothenate biosynthesis, with implications for bacterial survival and symbiotic efficiency. Comprehensive transcriptomic and metabolomic analyses have revealed distinct responses to various environmental challenges:

To methodologically investigate these responses, researchers should:

• Employ quantitative RT-PCR to measure transcript levels of both panC genes under controlled stress conditions.

• Create dual reporter strains containing panC1p-GFP and panC2p-mCherry to visualize differential expression patterns.

• Use LC-MS/MS to quantify intracellular pantothenate and CoA levels under various stressors.

• Apply 13C-labeling and metabolic flux analysis to track carbon flow through the pantothenate pathway during stress responses.

• Generate panC2 overexpression and knockout strains to assess stress tolerance phenotypes.

These findings suggest that panC2 may function as a stress-responsive isoform that ensures continued pantothenate synthesis under adverse conditions, potentially explaining the evolutionary maintenance of this gene duplication.

What approaches can be used to study the interaction network of PanC2 in Bradyrhizobium japonicum?

Understanding the protein-protein interaction network of PanC2 provides insights into its functional integration within bacterial metabolism and potential regulatory mechanisms. Methodologically, researchers can employ several complementary approaches:

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

  • Create strains expressing tagged PanC2 (e.g., His-tag, FLAG-tag)

  • Perform pull-down experiments under various growth conditions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions through reciprocal pull-downs

• Bacterial two-hybrid screening:

  • Use PanC2 as bait against a B. japonicum genomic library

  • Screen for positive interactions on selective media

  • Sequence positive clones to identify interaction partners

  • Verify interactions through co-immunoprecipitation

• Protein proximity labeling:

  • Generate a PanC2-BioID fusion protein

  • Allow in vivo biotinylation of proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

Interactome studies have revealed several functional interaction partners:

These interaction studies suggest that PanC2 may function as part of a multienzyme complex that coordinates pantothenate synthesis with broader metabolic processes and symbiotic functions. The differential interaction profiles of PanC1 and PanC2 further support the hypothesis of functional specialization following gene duplication.