Recombinant Bradyrhizobium japonicum Phosphoribosylformylglycinamidine synthase 2 (purL), partial

What is the biochemical function of Phosphoribosylformylglycinamidine synthase in Bradyrhizobium japonicum?

Phosphoribosylformylglycinamidine synthase (EC 6.3.5.3) catalyzes a critical step in de novo purine biosynthesis. The enzyme specifically catalyzes the conversion of N2-formyl-N1-(5-phospho-D-ribosyl)glycinamide, ATP, and glutamine to 2-(formamido)-N1-(5-phospho-D-ribosyl)acetamidine, ADP, phosphate, and glutamate . In B. japonicum, this enzyme plays an essential role in supporting bacterial growth and metabolism, which is particularly important during symbiotic nitrogen fixation with legume hosts such as soybean . The purine biosynthesis pathway is critical for the production of nucleotides required for DNA and RNA synthesis, as well as for other essential cellular processes during both free-living growth and symbiotic states.

How does the purL gene differ from other purine biosynthesis genes in B. japonicum?

The purL gene encodes Phosphoribosylformylglycinamidine synthase, which belongs to the carbon-nitrogen ligase family, specifically those forming carbon-nitrogen bonds with glutamine as amido-N-donor . Unlike other purine biosynthesis genes, purL exhibits distinctive expression patterns depending on environmental conditions and symbiotic states. In B. japonicum, purL is part of a network of genes necessary for purine metabolism that may show functional redundancy, similar to what has been observed with sulfonate utilization operons . The gene's expression is likely coordinated with other metabolic pathways critical for nitrogen fixation and symbiotic relationships with host plants.

What expression systems are most effective for producing recombinant B. japonicum purL?

For recombinant expression of B. japonicum purL, several systems have been successfully employed with varying efficiencies:

For optimal expression of soluble and functional protein, the E. coli Arctic Express system with induction at 16°C and supplementation with rare codons often provides the best balance between yield and proper folding. Addition of fusion tags such as MBP (maltose-binding protein) or SUMO can further enhance solubility, particularly for partial constructs that may lack stabilizing domains.

What are the optimal conditions for assaying recombinant B. japonicum purL activity?

Accurate determination of purL enzymatic activity requires careful optimization of assay conditions:

The enzyme is particularly sensitive to oxidation and metal ion contaminants. Inclusion of 10% glycerol and 0.1 mM EDTA in all buffers improves stability. For partial purL constructs, longer pre-incubation times (15-20 minutes) at the assay temperature may be necessary to achieve full activity due to slower equilibration kinetics.

How should site-directed mutagenesis be designed to investigate catalytic mechanisms of B. japonicum purL?

Site-directed mutagenesis provides critical insights into catalytic mechanisms and structure-function relationships. For B. japonicum purL, a systematic approach should target:

• Predicted glutamine binding site residues (typically conserved Cys, His, Asp catalytic triad)

• ATP binding site (conserved P-loop motif and magnesium coordination residues)

• Substrate binding pocket residues (based on homology with structural data from other species)

• Inter-domain communication residues (those at domain interfaces)

Conservative mutations (e.g., Asp→Glu, Lys→Arg) should be tested alongside more disruptive changes (e.g., charged→neutral, hydrophilic→hydrophobic). Assessment of mutant activity under standard conditions, complemented by substrate variation studies, provides comprehensive mechanistic insights. Circular dichroism spectroscopy should be used to confirm that mutations do not cause significant structural alterations that could indirectly affect activity.

What strategies can be used to determine if the partial B. japonicum purL retains full catalytic activity?

Determining whether a partial purL construct retains full catalytic activity requires a multi-faceted approach:

• Comparative kinetic analysis between partial and full-length enzyme:

  • Measure Km, kcat, and kcat/Km for all substrates

  • Determine inhibition constants for product and substrate analogs

  • Analyze pH and temperature dependence profiles

• Domain analysis:

  • Identify missing domains/regions in the partial construct

  • Assess potential impact on catalytic center and substrate binding

  • Consider effects on oligomerization if applicable

• Complementation studies:

  • Test whether the partial purL can complement growth defects in purL-deficient bacterial strains

  • Compare growth rates under various nutrient limitations

The partial enzyme will likely show altered parameters in at least some of these analyses, with the specific deficiencies providing insight into the functional roles of the missing regions.

How does sulfur metabolism in B. japonicum interact with purL function and expression?

B. japonicum demonstrates remarkable versatility in sulfur source utilization, including sulfate, cysteine, sulfonates, and sulfur-ester compounds for growth . This metabolic flexibility likely affects purL function through several mechanisms:

• Cysteine availability directly impacts protein folding and stability due to critical disulfide bonds or metal coordination sites in purL.

• Sulfur limitation may trigger regulatory responses that affect purine biosynthesis pathway genes, including purL, through:

  • Direct transcriptional regulation via sulfur-responsive transcription factors

  • Post-translational modifications affected by sulfur availability

  • Allosteric regulation by sulfur-containing metabolites

• B. japonicum exhibits specific sulfonate utilization gene clusters (bll6449-bll6455 and bll7007-bll7011) that are expressed during symbiotic nodulation , potentially coordinated with purL expression when the bacterium transitions to symbiotic states.

The strategic connection between sulfur metabolism and nitrogen fixation may involve purL regulation as a central component of nucleotide biosynthesis necessary for symbiotic processes. Tracking purL expression under different sulfur source conditions can reveal these regulatory relationships.

What role does purL play in B. japonicum symbiotic efficiency with legume hosts?

PurL likely plays several critical roles in establishing and maintaining efficient symbiotic relationships:

• During the initial infection and nodule formation stages, rapid bacterial proliferation requires high purine biosynthesis capacity for nucleic acid synthesis.

• The energy-intensive nitrogen fixation process in mature bacteroids demands efficient ATP utilization, requiring tight regulation of ATP-consuming enzymes like purL.

• Purine derivatives serve as important signaling molecules in plant-microbe interactions, potentially mediating aspects of the symbiotic relationship.

Research approaches to investigate these roles include:

• Constructing conditional purL mutants to evaluate effects on different symbiotic stages

• Quantifying purL expression throughout the symbiotic cycle using qRT-PCR

• Metabolomic analysis of purine pathway intermediates in free-living versus symbiotic states

• Comparing symbiotic efficiency of B. japonicum strains with various purL expression levels

Notably, B. japonicum exhibits functional redundancy in certain metabolic pathways , suggesting potential compensatory mechanisms may exist for purL function during symbiosis.

How can structural information be obtained for partial B. japonicum purL when crystallization is challenging?

When crystallization of partial purL proves difficult, alternative structural biology approaches include:

• Cryo-electron microscopy (cryo-EM):

  • Particularly valuable for larger protein complexes

  • Can achieve near-atomic resolution without crystallization

  • May reveal dynamic conformational states

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

  • Provides low-resolution envelope structures in solution

  • Can track conformational changes upon substrate binding

  • Complements computational modeling approaches

• Nuclear magnetic resonance (NMR) for domain analysis:

  • Suitable for individual domains under 25-30 kDa

  • Provides dynamic information not available from static structures

  • Can identify substrate binding sites through chemical shift perturbation

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

  • Maps protein flexibility and solvent accessibility

  • Identifies conformational changes upon substrate binding

  • Helps define domain boundaries and interactions

• Integrative computational modeling:

  • Combines homology modeling with experimental constraints

  • Molecular dynamics simulations to predict dynamic properties

  • Deep learning approaches for structure prediction (AlphaFold2, RoseTTAFold)

A combined approach utilizing multiple complementary techniques often provides the most comprehensive structural understanding.

How should contradictory kinetic data from purL activity assays be reconciled?

Contradictory kinetic data often stems from methodological variations or biochemical complexities of the enzyme. A systematic troubleshooting approach includes:

• Assay methodology analysis:

  • Compare direct versus coupled assay systems

  • Evaluate potential interference from detection components

  • Standardize enzyme storage and pre-incubation conditions

• Enzyme heterogeneity assessment:

  • Analyze protein oligomeric state by size-exclusion chromatography

  • Check for proteolytic degradation by SDS-PAGE

  • Verify post-translational modifications by mass spectrometry

• Substrate and cofactor effects:

  • Test for substrate inhibition at higher concentrations

  • Evaluate order of substrate addition effects

  • Identify potential allosteric regulators or contaminants

• Statistical analysis:

  • Use global fitting of datasets with multiple models

  • Apply Akaike Information Criterion to identify optimal kinetic models

  • Conduct experiments with sufficient replicates for meaningful statistical comparisons

For partial purL constructs, protein stability during the assay should be carefully monitored, as truncated proteins often exhibit time-dependent activity loss that can manifest as apparent kinetic inconsistencies.

What comparative genomics approaches can provide insights into B. japonicum purL function?

Comparative genomics offers powerful insights into functional conservation and specialization of purL:

This integrated approach can reveal specialized adaptations in B. japonicum purL related to its symbiotic lifestyle and identify potential regulatory elements governing expression during different physiological states. Careful attention to B. japonicum-specific codon usage and GC content biases is necessary when interpreting comparative data across diverse bacterial species.

What purification strategy yields the highest activity for recombinant B. japonicum purL?

A streamlined purification protocol optimized for activity retention involves:

• Cell lysis:

  • Gentle mechanical disruption (microfluidizer at 10,000-15,000 psi)

  • Buffer composition: 50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 1 mM PMSF

  • Inclusion of lysozyme (0.2 mg/mL) and DNase I (10 μg/mL)

• Initial capture:

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

  • Gradient elution (20-250 mM imidazole) to separate populations with different binding characteristics

• Intermediate purification:

  • Ion exchange chromatography (IEX) using Q-Sepharose at pH 7.5

  • Salt gradient (50-500 mM NaCl) to remove contaminants with similar IMAC profiles

• Polishing:

  • Size exclusion chromatography (Superdex 200) to resolve oligomeric states and remove aggregates

  • Buffer: 25 mM HEPES pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT

All purification steps should be performed at 4°C with minimal delay between steps. For partial purL constructs, inclusion of stabilizing additives (0.5 M arginine or 50 mM glutamate/glutamine) may significantly improve stability and recovery of active protein. Activity measurements should be performed after each step to track recovery and specific activity improvements.

How can the expression of recombinant B. japonicum purL be optimized in E. coli?

Optimization of heterologous expression requires addressing several parameters:

• Strain selection:

  • BL21(DE3) for high expression

  • Rosetta for rare codon supplementation

  • ArcticExpress for low-temperature expression

  • SHuffle for enhanced disulfide bond formation

• Codon optimization:

  • Adjust for E. coli codon bias while avoiding problematic mRNA secondary structures

  • Use harmonization rather than maximization approach to maintain translational pausing patterns

• Induction parameters:

  • Temperature: 16-18°C for improved folding

  • IPTG concentration: 0.1-0.2 mM for slower, more controlled expression

  • Induction timing: Mid-log phase (OD600 0.6-0.8)

  • Duration: Extended overnight expression at lower temperatures

• Media composition:

  • Auto-induction media for gradual protein production

  • Supplementation with trace elements, particularly zinc and magnesium

  • Addition of compatible solutes (betaine, proline) for osmotic assistance

For partial purL constructs that may lack stabilizing domains, co-expression with chaperones (GroEL/ES, DnaK/J/GrpE) can dramatically improve folding and soluble yield. Fusion to solubility-enhancing partners (MBP, SUMO, TrxA) with appropriate linker design is particularly effective for challenging partial constructs.

How might engineering B. japonicum purL affect nitrogen fixation efficiency in agricultural applications?

Strategic engineering of purL could potentially enhance B. japonicum's agricultural applications:

These approaches could lead to improved inoculant strains with enhanced nitrogen fixation capacity, reducing the need for chemical fertilizers in soybean and other legume crops.

What analytical techniques can best characterize the conformational dynamics of purL during catalysis?

Understanding conformational dynamics is crucial for elucidating catalytic mechanisms:

• Single-molecule FRET:

  • Strategic placement of fluorophore pairs to monitor domain movements

  • Real-time observation of conformational changes during catalysis

  • Detection of rare or transient conformational states

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

  • Mapping regions of altered solvent accessibility during the catalytic cycle

  • Identifying conformational changes upon substrate binding

  • Comparing dynamics between partial and full-length constructs

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Chemical shift perturbation to map ligand binding sites

  • Relaxation dispersion experiments to detect microsecond-millisecond motions

  • Residual dipolar coupling measurements to determine relative domain orientations

• Molecular dynamics simulations:

  • Integration with experimental data to model complete catalytic cycle

  • Prediction of rate-limiting conformational changes

  • Identification of potential allosteric sites

The combination of these approaches can reveal how substrate binding triggers conformational changes necessary for catalysis and how these dynamics might differ in the partial purL construct compared to the full-length enzyme.