Recombinant Rhodopirellula baltica Adenylosuccinate synthetase (purA)

What is the biological role of adenylosuccinate synthetase (purA) in Rhodopirellula baltica?

Adenylosuccinate synthetase (purA) in R. baltica likely plays a central role in purine nucleotide biosynthesis, similar to other bacterial species. This enzyme catalyzes the first committed step in the conversion of IMP to AMP by facilitating the reaction between IMP, GTP, and aspartate to form adenylosuccinate. In the marine bacterium R. baltica, this enzyme would be essential for growth and cellular functions, particularly during the exponential growth phase when DNA replication and RNA synthesis are highly active. The expression pattern of purA in R. baltica may correlate with different growth stages, as seen with many metabolic genes that show differential regulation throughout its life cycle .

What purification strategy should be employed for recombinant R. baltica purA?

A multi-step purification strategy is recommended for R. baltica purA:

• Affinity chromatography: Incorporate an N-terminal or C-terminal affinity tag (His6, GST, or MBP) in your expression construct for initial capture. Based on the experience with other R. baltica proteins, the recombinant purA should contain "an N-terminal tag and may also contain a C-terminal tag" .

• Ion exchange chromatography: As a second step, considering the theoretical pI of the protein to select appropriate resin and buffer conditions.

• Size exclusion chromatography: For final polishing and to ensure removal of aggregates or degradation products.

For buffer composition, a Tris-based buffer with 50% glycerol for storage has been successful for other recombinant proteins . Aim for a final purity of >85% as assessed by SDS-PAGE, which is standard for recombinant protein preparations used in research .

How should researchers address potential solubility issues with recombinant R. baltica purA?

When encountering solubility challenges with R. baltica purA, implement these methodological approaches:

• Optimize expression conditions: Test multiple temperatures (18°C, 25°C, 30°C), induction concentrations, and expression durations.

• Modify fusion tags: If initial constructs show poor solubility, experiment with solubility-enhancing tags such as MBP, SUMO, or Trx.

• Add stabilizing agents to lysis buffers: Include osmolytes (glycerol, sucrose), mild detergents (0.1% Triton X-100), or stabilizing salts. R. baltica naturally accumulates compatible solutes like mannosylglucosylglycerate (MGG) that may enhance protein stability .

• Consider the unique physiology of R. baltica: As a marine organism with salt resistance , incorporation of moderate salt concentrations (100-300 mM NaCl) in buffers may improve protein stability and solubility.

• Evaluate co-expression with chaperones: If necessary, co-express with molecular chaperones to facilitate proper folding.

Monitor the effect of these interventions using activity assays alongside SDS-PAGE analysis to ensure that soluble protein is properly folded and enzymatically active.

What is the most reliable method for measuring adenylosuccinate synthetase activity from R. baltica?

The most reliable method for measuring R. baltica purA activity is a spectrophotometric assay that monitors the formation of adenylosuccinate from IMP, aspartate, and GTP. The reaction can be followed by:

• Direct measurement: Monitor the increase in absorbance at 280 nm due to the formation of adenylosuccinate.

• Coupled enzyme assay: Use auxiliary enzymes to couple adenylosuccinate formation to NADH oxidation, which can be monitored at 340 nm.

• HPLC analysis: Separate and quantify reaction substrates and products.

For optimal activity, the assay buffer should include Mg²⁺ as a cofactor, as this enzyme typically requires divalent cations. Based on experience with other R. baltica enzymes, researchers should verify enzyme activity in cell extracts before proceeding with recombinant protein characterization . When determining kinetic parameters, test a range of substrate concentrations to construct accurate Michaelis-Menten plots. The specific activity for purified recombinant purA should be reported in μmol/min·mg, similar to how activity was reported for other R. baltica enzymes (e.g., MggB had a specific activity of 0.013 ± 0.1 μmol/min·mg) .

How does salt concentration affect purA activity and stability, considering R. baltica's marine origin?

Given R. baltica's adaptation to marine environments with "salt resistance" , its purA likely exhibits distinct responses to salt concentration:

To experimentally determine optimal salt conditions, conduct activity assays across a salt gradient (0-1000 mM NaCl) and assess both initial activity and stability over time. Previous studies with R. baltica show it can adapt to varying salt conditions, suggesting its enzymes may have evolved mechanisms to function across a range of salt concentrations . Additionally, test whether other ions (K⁺, Mg²⁺) have specific effects on activity, as these ions often play important roles in maintaining enzyme structure and function in marine organisms.

How can researchers investigate the substrate specificity of R. baltica purA to identify potential novel functions?

To thoroughly investigate substrate specificity of R. baltica purA:

• Compare canonical substrates: First establish baseline activity with standard substrates (IMP, GTP, and aspartate).

• Test nucleotide analogs: Systematically substitute structural analogs of IMP to identify the key recognition elements.

• Evaluate alternative phosphate donors: Replace GTP with other nucleoside triphosphates (ATP, UTP, CTP) to determine specificity.

• Assess amino acid preference: Test aspartate analogs and other amino acids to define the amino acid binding pocket specificity.

• Investigate temperature and pH dependencies: R. baltica's adaptations to marine environments may have resulted in unique temperature and pH optima compared to mesophilic counterparts.

• Conduct comparative analysis: Compare kinetic parameters with purA from other species, particularly focusing on the kcat/Km ratio as an indicator of catalytic efficiency.

This methodical approach may reveal specialized adaptations in R. baltica purA that reflect its evolutionary history within the deep-branching Planctomycetes phylum and its adaptation to marine ecological niches.

What structural features distinguish R. baltica purA from other bacterial adenylosuccinate synthetases?

While specific structural information for R. baltica purA isn't provided in the search results, researchers can anticipate several distinguishing features based on R. baltica's evolutionary position and marine adaptation:

• Salt-bridge networks: Being a marine bacterium with demonstrated salt resistance , R. baltica purA likely contains an increased number of surface salt bridges to maintain stability in high-salt environments.

• Domain architecture: The enzyme likely maintains the three-domain architecture typical of adenylosuccinate synthetases, but may contain unique loops or insertions, particularly in surface-exposed regions.

• Substrate binding pocket: Potential adaptations in the binding pocket may reflect specificity for substrates available in R. baltica's native environment.

To elucidate these features, researchers should pursue:

• X-ray crystallography or cryo-EM studies to determine high-resolution structures

• Molecular dynamics simulations to understand conformational dynamics, especially under varying salt conditions

• Homology modeling based on related structures, with careful validation of model quality

Comparative analysis with structures like the ModBase 3D structure referenced for the Brucella abortus purA (P0C115) would provide valuable insights into R. baltica-specific adaptations.

How can site-directed mutagenesis be effectively used to probe the active site of R. baltica purA?

A systematic site-directed mutagenesis approach for R. baltica purA should target:

• Putative catalytic residues: Based on sequence alignments with well-characterized adenylosuccinate synthetases, identify and mutate conserved residues likely involved in catalysis (typically acidic residues that coordinate metal ions or basic residues involved in substrate binding).

• Substrate-binding residues: Mutate residues predicted to interact with IMP, GTP, and aspartate.

• Allosteric sites: Investigate potential regulatory sites by creating mutations in regions away from the active site.

For each mutant:

• Verify proper folding using circular dichroism and thermal shift assays

• Determine kinetic parameters (kcat, Km) for all substrates

• Assess protein stability under various conditions

This approach will not only elucidate the catalytic mechanism but may also reveal unique features of R. baltica purA that reflect its adaptation to marine environments. When designing mutations, consider the expression challenges observed with other R. baltica proteins, where removal of additional sequences was necessary to obtain active protein .

What computational approaches are most suitable for predicting protein-protein interactions involving R. baltica purA?

For predicting protein-protein interactions (PPIs) involving R. baltica purA, researchers should employ a multi-faceted computational approach:

• Sequence-based methods:

  • Search for conserved interaction motifs

  • Use co-evolution analysis to identify potential binding partners

  • Apply machine learning algorithms trained on known bacterial PPIs

• Structure-based methods:

  • Perform protein-protein docking simulations

  • Analyze surface electrostatics and hydrophobicity to identify potential interaction interfaces

  • Use template-based modeling if structures of homologous complexes are available

• Network-based approaches:

  • Integrate gene expression data from different growth phases of R. baltica

  • Analyze genomic context (gene neighborhood, fusion events)

  • Consider differential expression patterns during R. baltica's life cycle phases

• Experimental validation design:

  • Plan pull-down assays with recombinant tagged purA

  • Design cross-linking experiments followed by mass spectrometry

  • Prepare bacterial two-hybrid system constructs

This comprehensive approach is particularly important for R. baltica proteins, as this organism has "an intriguing lifestyle and cell morphology" that may involve unique protein interaction networks compared to model organisms.

How can recombinant R. baltica purA contribute to understanding evolutionary adaptations in the Planctomycetes phylum?

Recombinant R. baltica purA offers a valuable tool for evolutionary studies of the Planctomycetes phylum:

• Comparative enzymology: By determining the kinetic parameters, substrate specificity, and stability profiles of R. baltica purA and comparing them with homologs from other bacterial phyla, researchers can identify Planctomycetes-specific adaptations. R. baltica is "a representative of the globally distributed phylum Planctomycetes" , making its enzymes important models for understanding this unique bacterial lineage.

• Structural biology insights: Structural studies of R. baltica purA can reveal adaptive features that evolved in response to the marine environment. These adaptations may include modifications to surface residues, altered flexibility of catalytic loops, or unique substrate binding determinants.

• Systems biology integration: Analyzing purA in the context of R. baltica's metabolic network, particularly during different growth phases where "numerous genes with potential biotechnological applications were found to be differentially regulated" , can elucidate how central metabolism has evolved in this phylum.

• Horizontal gene transfer assessment: Phylogenetic analysis of purA sequences can help identify potential horizontal gene transfer events that shaped Planctomycetes evolution.

This research would contribute to our understanding of "deep-branching" bacterial phyla and how metabolic enzymes adapt to specialized ecological niches.

What are the methodological considerations for using R. baltica purA in structural biology studies?

When pursuing structural studies of R. baltica purA, researchers should consider these methodological approaches:

• Protein preparation:

  • Optimize protein expression to yield milligram quantities of homogeneous protein

  • Ensure >95% purity by implementing rigorous purification protocols

  • Verify protein monodispersity by dynamic light scattering

  • Screen buffer conditions to maximize stability during concentration

• Crystallization strategies:

  • Perform extensive crystallization screening (1000+ conditions)

  • Consider crystallizing with substrates, products, or substrate analogs

  • Test both full-length protein and engineered constructs with flexible regions removed

  • Include salt concentrations reflecting R. baltica's marine environment

• Cryo-EM considerations:

  • Evaluate protein size (adenylosuccinate synthetases are typically ~45-50 kDa, which is challenging for cryo-EM)

  • Consider forming complexes with antibody fragments to increase size

  • Optimize grid preparation to prevent preferred orientation

• NMR approaches:

  • For dynamics studies, prepare isotopically labeled protein

  • Focus on specific domains or binding interactions rather than full-length protein

  • Design experiments to probe salt-dependent conformational changes

• Data analysis:

  • Perform comparative analysis with structures from diverse bacterial phyla

  • Analyze surface properties in the context of marine adaptation

  • Examine oligomerization states under different conditions

Integrating these approaches will provide valuable insights into R. baltica purA structure and function relationships.

How can knowledge of R. baltica purA inform the development of novel antimicrobials targeting Planctomycetes-related pathogens?

While Planctomycetes are not typically pathogenic, insights from R. baltica purA can inform antimicrobial development strategies:

• Identification of unique structural features: Detailed comparison of R. baltica purA with homologs from other bacterial phyla can reveal Planctomycetes-specific features that could be exploited for selective inhibition. These unique elements might be found in the active site or in allosteric regulatory regions.

• Rational inhibitor design: Based on structural and biochemical characterization, design inhibitors that:

  • Target conserved residues within Planctomycetes but differ from other bacterial phyla

  • Exploit unique substrate binding preferences

  • Take advantage of distinct conformational dynamics

• High-throughput screening strategies:

  • Develop efficient activity assays for screening compound libraries

  • Use computational docking against the R. baltica purA structure to identify potential leads

  • Implement fragment-based approaches to develop high-affinity inhibitors

• Selectivity assessment: Compare inhibition profiles across purA enzymes from different bacterial phyla to ensure selectivity for Planctomycetes-related targets.

• Resistance mechanism prediction: Analyze the genetic variability of purA within Planctomycetes to predict potential resistance mechanisms and design inhibitors that remain effective despite these variations.

This approach leverages R. baltica's position as "a model organism" within Planctomycetes to develop broader strategies applicable to related organisms of potential clinical interest.

What are the common pitfalls in expressing and purifying R. baltica proteins, and how can they be addressed?

Based on experiences with other R. baltica proteins, researchers should anticipate and address these common challenges:

• Incorrect annotation of start codons: As observed with the GpgS gene where "no activity was detected for this recombinant protein" until the correct start codon was identified 240 bp downstream , carefully analyze sequence data to identify the true coding region. Solution: Design multiple constructs with different start sites and test for activity.

• Protein solubility issues: Marine proteins often have distinct folding properties. Solution: Screen multiple expression conditions (temperature, induction level) and include solubility-enhancing tags (MBP, SUMO). Consider adding osmolytes like glycerol, which is used in storage buffers for other recombinant proteins .

• Protein instability during purification: R. baltica proteins may require specific conditions for stability. Solution: Include protease inhibitors during lysis and minimize purification time. Include glycerol in all buffers, as seen in the storage buffer recommendation for other recombinant proteins: "Tris-based buffer, 50% glycerol" .

• Loss of activity during purification: Some R. baltica enzymes like MggB showed that "attempts to further improve the purity of the protein led to the loss of activity" . Solution: Optimize purification steps to balance purity with activity retention, and consider keeping stabilizing factors throughout purification.

• Tag interference with activity: N-terminal or C-terminal tags may affect enzyme function. Solution: Test both N and C-terminal tag positions and include tag removal options via protease cleavage sites.

By anticipating these challenges, researchers can develop more effective expression and purification strategies for R. baltica purA.

How should researchers approach the design of kinetic experiments to account for R. baltica's unique physiological conditions?

When designing kinetic experiments for R. baltica purA, researchers should incorporate the following methodological considerations:

• Buffer composition optimization:

  • Test buffers that mimic marine conditions (elevated salt concentrations)

  • Include divalent cations (Mg²⁺, Mn²⁺) at physiologically relevant concentrations

  • Assess the effect of compatible solutes like mannosylglucosylglycerate (MGG), which R. baltica naturally accumulates

• Temperature considerations:

  • Perform assays at a range of temperatures (15-40°C) to determine optimal activity

  • Conduct thermal stability assays to understand temperature-dependent unfolding

  • Consider R. baltica's marine habitat when interpreting temperature effects

• Experimental design for kinetic analysis:

  • Use initial velocity measurements under conditions where <10% of substrate is consumed

  • Vary one substrate while keeping others at saturating concentrations

  • Include proper controls for background reactions and spontaneous substrate degradation

• Data analysis approach:

  • Fit data to appropriate enzyme kinetic models (Michaelis-Menten, Hill, etc.)

  • Use global fitting approaches when analyzing bi-substrate kinetics

  • Apply statistical tests to validate the selected kinetic model

• Physiological relevance assessment:

  • Connect enzyme properties to R. baltica's growth phases

  • Consider how enzyme activity might change during the transition from swarmer cells to rosette formation

This comprehensive approach will yield kinetic parameters that accurately reflect the behavior of purA in R. baltica's native physiological context.

What strategies can enhance the reproducibility of experiments with recombinant R. baltica purA across different laboratories?

To maximize experimental reproducibility with recombinant R. baltica purA across laboratories:

• Standardize expression constructs and protocols:

  • Share complete plasmid sequences including vector backbones and all fusion tags

  • Specify exact expression conditions (strain, media composition, temperature, induction parameters)

  • Document all buffer compositions precisely, including pH adjustment method

• Implement robust quality control measures:

  • Define minimum purity standards (e.g., >85% by SDS-PAGE as used for other recombinant proteins)

  • Establish activity benchmarks using standard assay conditions

  • Develop and share spectroscopic profiles (UV-Vis, fluorescence, CD) of properly folded protein

• Create detailed methodology for activity assays:

  • Specify exact reaction conditions, including temperature control methods

  • Define data collection parameters (time points, measurement intervals)

  • Share raw data processing workflows and analysis scripts

• Address protein stability and storage:

  • Establish standard storage conditions (e.g., "Tris-based buffer, 50% glycerol" at "-20 degrees C")

  • Define maximum storage duration with activity retention data

  • Document freeze-thaw stability and recommend aliquoting strategies

• Implement collaborative validation:

  • Establish round-robin testing between laboratories

  • Create reference material batches that can be distributed

  • Develop benchmarking datasets for key experimental outcomes

These approaches will significantly enhance reproducibility and facilitate meaningful comparison of results across research groups working with this challenging but scientifically valuable enzyme system.