Recombinant Geobacter sulfurreducens Adenylosuccinate synthetase (purA)

What is Adenylosuccinate synthetase and its role in G. sulfurreducens metabolism?

Adenylosuccinate synthetase (AdSS) is an essential enzyme in purine metabolism that catalyzes the first step in the conversion of IMP to AMP. In bacteria like G. sulfurreducens, AdSS functions at a regulatory point in purine metabolism, making it critical for cellular growth and survival. The enzyme catalyzes the reaction: IMP + aspartate + GTP → adenylosuccinate + GDP + Pi, serving as a key component of the purine salvage pathway .

G. sulfurreducens, like many bacteria, depends on efficient nucleotide synthesis for growth, especially in its unique anaerobic respiratory environments. The purA gene and its encoded protein are likely essential for G. sulfurreducens, particularly when considering its adaptability to different electron acceptors such as Fe(III) oxides and electrode surfaces.

How does purA deletion affect G. sulfurreducens growth compared to other gene deletions?

While specific data on purA deletion in G. sulfurreducens is limited in these search results, we can draw parallels from other deletion studies. Unlike the pgcA deletion, which specifically impairs Fe(III) and Mn(IV) oxide reduction while maintaining electrode respiration capability , a purA deletion would likely have more fundamental metabolic consequences.

Studies in other bacteria have shown that purA mutants are typically adenine or adenosine auxotrophs, requiring these compounds for growth. For example, in Helicobacter pylori, a purA mutant "is capable of growth only in a medium supplemented with adenine or adenosine and that its growth was significantly retarded in nutrient-rich medium, thus indicating the essentiality of AdSS for the salvage pathway" .

The established markerless deletion methods used for other G. sulfurreducens genes (as described for pgcA ) would be applicable for creating a purA deletion strain for comparative studies.

What is the recommended protocol for cloning and expressing recombinant G. sulfurreducens purA?

Based on successful approaches with similar bacterial enzymes, the following protocol is recommended:

• PCR Amplification of the purA Gene:

  • Design primers with appropriate restriction sites (e.g., NdeI and XhoI) for directional cloning

  • Use high-fidelity polymerase with genomic DNA template from G. sulfurreducens

  • Recommended PCR conditions: initial denaturation at 98°C for 30s, followed by 30 cycles of (98°C for 10s, 60°C for 30s, 72°C for 1min), and final extension at 72°C for 10min

• Cloning into Expression Vector:

  • Digest the PCR product and pET21b vector with NdeI and XhoI

  • Ligate and transform into E. coli cloning strain

  • Verify the construct by sequencing

• Expression in E. coli:

  • Transform the confirmed plasmid into E. coli BL21-CodonPlus(DE3)-RIL

  • Grow cultures to OD600 = 0.6 at 37°C

  • Induce with 0.5mM IPTG

  • Continue growth for 4 hours

  • Harvest cells by centrifugation at 5000×g at 4°C

This approach parallels the successful expression of H. pylori AdSS and can be adapted for G. sulfurreducens purA.

What purification strategy yields the highest purity and activity for recombinant G. sulfurreducens AdSS?

A three-step chromatographic purification procedure is recommended:

• Initial Extraction:

  • Lyse cells in phosphate buffer (pH 6.5) containing 2mM EDTA, 0.1mM PMSF, and 1mM DTT

  • Use lysozyme (1mg/mL) treatment followed by sonication

  • Add DNaseI (3.6μg/mL) to reduce viscosity

  • Clarify by centrifugation at 50,000×g

• Chromatographic Purification:

  • Ion exchange chromatography (DEAE or S-Sepharose depending on the theoretical pI)

  • Hydrophobic interaction chromatography (Phenyl-Sepharose)

  • Size exclusion chromatography (Superdex 200) for final polishing

• Activity Verification:

  • Monitor enzymatic activity spectrophotometrically at 280nm

  • Reaction mixture should contain IMP, GTP, aspartate, and Mg2+

  • Calculate activity using extinction coefficient 1.17×104 M−1cm−1

This purification strategy maintains protein stability while removing contaminants that might interfere with structural or kinetic studies.

How can the activity and stability of G. sulfurreducens AdSS be optimized for in vitro studies?

Optimizing AdSS activity requires careful consideration of several factors:

• Buffer Composition:

  • Use phosphate buffer (50mM) with pH optimized between 6.5-7.5

  • Include Mg2+ (essential cofactor, typically 5-10mM)

  • Add stabilizing agents: 1-2mM DTT to maintain reduced cysteines

  • Consider including 5-10% glycerol to enhance stability

• Temperature and pH Optimization:

  • Determine optimal temperature (likely 30-37°C based on G. sulfurreducens growth conditions)

  • Test narrow pH range (6.5-7.5) to identify optimal enzymatic activity

  • Consider G. sulfurreducens' natural environment when designing stability tests

• Storage Conditions:

  • Store purified enzyme at -80°C with 20% glycerol

  • Avoid repeated freeze-thaw cycles

  • For working stocks, maintain at 4°C with reducing agents

Testing multiple conditions in parallel allows rapid identification of optimal parameters for maximum activity retention.

How does AdSS activity in G. sulfurreducens compare with other bacteria, particularly in relation to electron acceptor availability?

G. sulfurreducens has a remarkable ability to utilize various electron acceptors, including insoluble metal oxides, soluble Fe(III) complexes, and electrode surfaces . This metabolic versatility may influence AdSS activity and regulation.

Comparative Analysis:

Research questions to address:

• Does AdSS activity differ when G. sulfurreducens is grown with different electron acceptors?

• Are there post-translational modifications of AdSS related to the redox state of the cell?

• How does AdSS expression change during adaptation to different growth conditions?

These investigations would require enzyme activity assays under various growth conditions and Western blot analysis to monitor expression levels.

What structural features distinguish G. sulfurreducens AdSS from homologs in other bacteria?

While specific structural data for G. sulfurreducens AdSS is not available in the search results, structural predictions and comparative analyses can provide valuable insights:

• Domain Architecture Analysis:

  • Bacterial AdSS typically exists as a homodimer

  • Each monomer consists of three domains: a GDP-binding N-terminal domain, a central domain with the active site, and a C-terminal domain involved in dimerization

  • Sequence analysis would reveal conservation of catalytic residues

• Homology Modeling Approach:

  • Use crystallized bacterial AdSS structures as templates

  • Pay particular attention to active site architecture

  • Analyze surface electrostatics for unique features

• Evolutionary Conservation:

  • Multiple sequence alignment across Geobacteraceae

  • Identification of conserved versus variable regions

  • Correlation with metabolic adaptations

This structural understanding would guide site-directed mutagenesis studies to identify residues critical for catalysis or substrate specificity.

How do kinetic parameters of G. sulfurreducens AdSS compare across different growth conditions?

The kinetic characterization of G. sulfurreducens AdSS would likely reveal adaptations to its unique metabolism:

Expected Experimental Design:

• Growth Condition Variables:

  • Electron acceptor type (Fe(III) oxide, Fe(III) citrate, electrode)

  • Carbon source (acetate vs. lactate)

  • Growth phase (early log, late log, stationary)

• Kinetic Parameters to Measure:

  • Km for IMP, aspartate, and GTP

  • kcat and catalytic efficiency (kcat/Km)

  • Inhibition constants for product inhibition

• Analytical Methods:

  • Steady-state kinetics using spectrophotometric assays

  • Isothermal titration calorimetry for binding studies

  • Potential regulatory metabolites as effectors

This comprehensive kinetic analysis would provide insights into how AdSS activity is modulated in response to the cell's energy status and environmental conditions.

How can researchers address protein stability issues when working with recombinant G. sulfurreducens AdSS?

Bacterial AdSS enzymes can present stability challenges during purification and characterization. The following strategies address common issues:

• Expression Optimization:

  • Lower induction temperature (16-25°C) to enhance proper folding

  • Co-expression with molecular chaperones

  • Use of solubility tags (MBP, SUMO) if necessary

• Stability Enhancement:

  • Buffer optimization with stabilizing agents (glycerol, trehalose)

  • Addition of ligands (IMP, GTP) during purification

  • Limited proteolysis to identify stable core domains

• Analytical Approaches:

  • Differential scanning fluorimetry to identify stabilizing conditions

  • Size exclusion chromatography with multi-angle light scattering to monitor oligomeric state

  • Circular dichroism to assess secondary structure integrity

For particularly difficult preparations, in vitro translation systems or alternative expression hosts like Shewanella oneidensis (which has been successful for other G. sulfurreducens proteins ) could be considered.

What approaches can resolve discrepancies in activity measurements for G. sulfurreducens AdSS?

When encountering inconsistent activity data, a systematic troubleshooting approach is essential:

• Method Validation:

  • Use multiple assay methods (spectrophotometric, HPLC-based, coupled enzyme)

  • Establish standard curves with known enzyme amounts

  • Include positive controls (commercially available AdSS)

• Variable Identification:

  • Test component stability (substrate degradation)

  • Examine buffer composition effects (ionic strength, pH)

  • Evaluate the presence of inhibitory contaminants

• Technical Considerations:

  • Ensure complete removal of imidazole after His-tag purification

  • Check for metal ion contamination affecting activity

  • Verify enzyme concentration with multiple methods

• Data Analysis:

  • Apply appropriate statistical tests for significance

  • Use non-linear regression for accurate kinetic parameter determination

  • Consider global fitting for complex kinetic mechanisms

This systematic approach helps identify sources of variability and establish reproducible assay conditions.

How can metabolomics approaches be integrated with purA studies in G. sulfurreducens?

Metabolomics provides valuable context for understanding AdSS function within the larger metabolic network:

• Experimental Design:

  • Compare wild-type vs. purA mutant (conditional if essential)

  • Analyze metabolite profiles under different electron acceptor conditions

  • Trace labeled precursors to quantify flux through purine pathways

• Analytical Platforms:

  • LC-MS/MS for comprehensive nucleotide and intermediate analysis

  • CE-MS for charged metabolites

  • NMR for structural confirmation and flux analysis

• Data Integration Approaches:

  • Correlation of AdSS activity with metabolite levels

  • Flux balance analysis incorporating enzyme kinetic data

  • Multi-omics integration with transcriptomics and proteomics

This integrated approach would reveal how AdSS activity influences and responds to the broader metabolic state, particularly in the context of G. sulfurreducens' unique respiratory versatility.

How might G. sulfurreducens AdSS be engineered for enhanced activity or altered specificity?

Protein engineering offers opportunities to modify AdSS properties for fundamental research or biotechnological applications:

• Rational Design Approaches:

  • Site-directed mutagenesis of substrate-binding residues

  • Introduction of disulfide bonds for enhanced stability

  • Engineering allosteric regulation sites

• Directed Evolution Strategies:

  • Development of selection systems based on adenine auxotrophy

  • High-throughput screening for activity under various conditions

  • Combinatorial approaches to identify beneficial mutations

• Application-Focused Modifications:

  • Creating temperature-stable variants

  • Engineering pH tolerance for broader operational range

  • Modifying substrate specificity for novel nucleotide analogs

These approaches would build on successful engineering of other purine metabolic enzymes while addressing the specific requirements of G. sulfurreducens metabolism.

What is the relationship between electron transfer capabilities and nucleotide metabolism in G. sulfurreducens?

G. sulfurreducens possesses remarkable extracellular electron transfer capabilities, which may interact with central metabolic pathways including nucleotide synthesis:

• Research Questions:

  • Does electron acceptor availability affect purine metabolism regulation?

  • How does energy status from different electron acceptors influence AdSS activity?

  • Is there a connection between cytochrome expression and nucleotide synthesis?

• Experimental Approaches:

  • Compare AdSS activity in cells grown with different electron acceptors

  • Examine purA expression during adaptation to new electron acceptors

  • Investigate metabolic flux changes when switching electron acceptors

• Theoretical Framework:

  • Energy conservation efficiency with different electron acceptors

  • NADH/NAD+ ratio effects on nucleotide synthesis

  • ATP availability impact on AdSS function

This research area would connect G. sulfurreducens' distinctive respiratory capabilities with fundamental metabolic processes.

How can systems biology approaches enhance our understanding of purA function in G. sulfurreducens?

Systems biology provides a framework to understand AdSS within the context of the entire cellular network:

• Genome-Scale Metabolic Modeling:

  • Incorporating AdSS kinetic parameters into constraint-based models

  • Predicting metabolic outcomes of purA mutations or altered expression

  • Simulating metabolic flux under various environmental conditions

• Network Analysis Approaches:

  • Identifying metabolic bottlenecks connected to AdSS activity

  • Examining regulatory networks governing purA expression

  • Determining synthetic lethal interactions with purA

• Multi-Omics Integration:

  • Correlating transcriptomics, proteomics, and metabolomics data

  • Constructing regulatory networks from multi-level data

  • Developing predictive models of adaptive responses

These approaches would place AdSS within its proper cellular context and reveal emergent properties not evident from studying the enzyme in isolation.