Recombinant Lactobacillus plantarum Adenylosuccinate synthetase (purA)

What is adenylosuccinate synthetase (purA) and what is its role in Lactobacillus plantarum?

Adenylosuccinate synthetase (purA) is an essential enzyme in the de novo pathway of purine nucleotide biosynthesis. In Lactobacillus plantarum, as in other organisms, it catalyzes the first committed step in the biosynthesis of adenosine monophosphate (AMP) from inosine monophosphate (IMP) . This critical metabolic reaction involves the conversion of IMP to adenylosuccinate using aspartate and GTP as substrates. The purA gene in L. plantarum (strain ATCC BAA-793 / NCIMB 8826 / WCFS1) encodes a protein of 429 amino acids with a molecular weight of approximately 47.2 kDa and belongs to the adenylosuccinate synthetase family .

The enzymatic reaction catalyzed by purA can be represented as:
IMP + Aspartate + GTP → Adenylosuccinate + GDP + Pi

This reaction requires magnesium ions as a cofactor and is energetically driven by GTP hydrolysis. The significance of purA in L. plantarum extends beyond basic metabolism, as purine nucleotides are essential components of DNA, RNA, and various metabolic cofactors required for bacterial growth and adaptation to different environmental conditions.

What expression systems are most effective for recombinant purA production in L. plantarum?

Based on successful expression of other recombinant proteins in Lactobacillus species, several expression systems can be effective for purA production. The pSIP expression system has proven particularly effective for heterologous protein production in L. plantarum . This system is regulated by a quorum-sensing mechanism that responds to a peptide pheromone, allowing tight control of gene expression.

For membrane-bound or metabolic proteins in L. plantarum, a detailed procedure using the pSIP system has been described, resulting in high yields of purified protein . When selecting an expression system for purA, researchers should consider:

• Promoter strength and inducibility: The pSIP system typically employs the strong, inducible promoters PsppA or PsppQ

• Vector features: Appropriate selection markers, origin of replication compatible with L. plantarum

• Fusion tags: N- or C-terminal tags to facilitate purification (His-tag, GST, etc.)

• Signal peptides: If secretion of the protein is desired

The effectiveness of the expression system can vary depending on the specific strain of L. plantarum used. Therefore, screening different host strains may be necessary to identify the optimal combination of expression vector and host strain for maximum purA yield.

How does L. plantarum compare to E. coli as an expression host for recombinant purA?

While E. coli remains the most commonly used host for recombinant protein expression, L. plantarum offers several advantages that make it an attractive alternative for purA expression:

• Safety profile: L. plantarum has GRAS (Generally Recognized As Safe) status, making it advantageous for applications where the safety of the expression host is a concern .

• Protein folding capabilities: L. plantarum may provide a more suitable environment for the correct folding of certain proteins that do not fold properly in E. coli. This is particularly evident in membrane proteins, where L. plantarum has demonstrated superior expression compared to E. coli .

• Native environment: For studying L. plantarum enzymes, expression in the native host eliminates potential issues with codon usage bias, protein folding, or post-translational modifications.

• Probiotic potential: L. plantarum has natural probiotic characteristics, which could be beneficial for certain applications .

A comparative study of RseP (a membrane-bound site-2-protease) expression demonstrated that L. plantarum yielded protein with higher purity compared to E. coli . When RseP was expressed in E. coli, detection of soluble protein failed in two of the three strains tested, whereas expression in L. plantarum was successful .

What are the critical parameters to optimize for successful expression of recombinant purA in L. plantarum?

Successful expression of recombinant purA in L. plantarum requires optimization of several critical parameters:

• Growth medium composition: The nutritional environment significantly impacts protein expression levels. For L. plantarum, media components such as carbon sources, nitrogen sources, and trace elements should be optimized. Research has shown that specific components like Na₂HPO₄, inulin, casein peptone, and certain amino acids (e.g., leucine) can significantly affect recombinant protein production in L. plantarum .

• Induction conditions: For inducible expression systems like pSIP:

  • Induction timing (typically during early to mid-logarithmic phase)

  • Inducer concentration

  • Duration of expression after induction

• Growth parameters:

  • Temperature: Often lower temperatures (20-25°C) after induction can improve protein folding

  • pH: L. plantarum is naturally acid-tolerant, growing well at pH values as low as 3.7

  • Aeration: As L. plantarum is microaerophilic, oxygen levels should be controlled

• Strain selection: Different strains of L. plantarum exhibit varying capabilities for heterologous protein expression. Strains isolated from specific environments may have advantageous properties for recombinant protein production .

• Vector design: Codon optimization for L. plantarum, appropriate promoter selection, and inclusion of effective ribosome binding sites can enhance expression levels.

A systematic approach using design of experiments (DOE) methodology, as employed for optimizing cell envelope proteinase production in L. plantarum LP69, can be effective for identifying optimal conditions for purA expression .

What purification strategies are most effective for recombinant purA from L. plantarum?

Effective purification of recombinant purA from L. plantarum typically involves multiple chromatographic steps, with the specific strategy depending on the expression construct and the required purity. A generalized purification workflow includes:

• Cell lysis: Efficient cell disruption is crucial for releasing intracellular proteins. L. plantarum has a more resistant cell wall compared to E. coli, often requiring stronger lysis conditions. Methods include:

  • Mechanical disruption (sonication, bead beating, high-pressure homogenization)

  • Enzymatic lysis (lysozyme treatment)

  • Chemical lysis with detergents

• Initial capture: Immobilized metal affinity chromatography (IMAC) is highly effective if the recombinant purA contains a His-tag. For a recombinant membrane protein expressed in L. plantarum, IMAC has been shown to be effective as an initial purification step .

• Intermediate purification: Ion exchange chromatography based on the theoretical isoelectric point of purA can be used to remove contaminants with different charge properties.

• Polishing: Size exclusion chromatography (SEC) for final purification and assessment of oligomeric state. This technique has been successfully used for purification of membrane proteins expressed in L. plantarum .

A purification workflow involving IMAC followed by SEC has yielded approximately 1 mg of pure protein per 3 g of wet-weight cell pellet for membrane proteins expressed in L. plantarum . For purA, which is a soluble cytoplasmic protein, yields could potentially be higher.

Buffer conditions during purification should be optimized to maintain enzyme stability and activity. Typical considerations include:

• pH (usually 7.0-8.0)

• Salt concentration

• Addition of stabilizing agents (glycerol, reducing agents)

• Protease inhibitors

How can the enzymatic activity of purified recombinant purA be accurately assessed?

Accurate assessment of purA enzymatic activity is essential for characterizing the recombinant enzyme. Several complementary methods can be employed:

• Spectrophotometric assays: The formation of adenylosuccinate from IMP and aspartate can be monitored by measuring absorbance changes. Adenylosuccinate has distinct spectral properties compared to the substrates.

• HPLC-based assays: High-performance liquid chromatography can be used to quantitatively separate and detect the substrates and products of the purA reaction. This approach has been used successfully for analyzing amino acids in recombinant L. plantarum strains .

• Coupled enzyme assays: The purA reaction can be linked to subsequent enzymatic reactions that produce easily detectable products, allowing for continuous monitoring of activity.

A typical reaction mixture for purA activity assessment includes:

• Purified purA enzyme (carefully quantified)

• IMP (substrate)

• Aspartate (substrate)

• GTP (energy source)

• Mg²⁺ (cofactor, typically as MgCl₂)

• Buffer system (often HEPES or Tris at pH 7.4-7.5)

The reaction kinetics can be determined by varying substrate concentrations and measuring initial reaction rates. From these data, important parameters such as Km (Michaelis constant) and kcat (turnover number) can be calculated to characterize the enzyme's catalytic efficiency.

For comparative studies, it is important to use standardized assay conditions and to include appropriate positive and negative controls. The specific activity of purA can be expressed as units of enzyme activity per milligram of protein (U/mg), where one unit is typically defined as the amount of enzyme that catalyzes the formation of 1 μmol of product per minute under defined conditions.

What structural characterization methods are suitable for recombinant L. plantarum purA?

Comprehensive structural characterization of recombinant L. plantarum purA requires a multi-technique approach:

For purA specifically, structural studies should focus on:

• The active site architecture

• Binding sites for IMP, aspartate, and GTP

• The coordination site for the essential Mg²⁺ cofactor

• Potential oligomerization interfaces

• Conformational changes during the catalytic cycle

By combining these complementary techniques, researchers can gain a comprehensive understanding of the structural properties of L. plantarum purA, which can inform functional studies and potential biotechnological applications.

How does purA activity contribute to acid stress tolerance in L. plantarum?

The potential role of purA in acid stress tolerance of L. plantarum can be understood through its connections to purine metabolism and cellular energetics. While direct evidence for purA's involvement in acid stress response is limited, parallels can be drawn from studies on related metabolic enzymes in Lactobacillus species.

Research on argininosuccinate synthase (ASS, encoded by argG), another enzyme involved in nucleotide metabolism, provides insights into how similar metabolic enzymes may contribute to acid tolerance. When the argG gene from Oenococcus oeni was heterologously expressed in L. plantarum, it significantly enhanced acid tolerance . The recombinant strain exhibited stronger growth performance under acidic conditions (pH 3.7) compared to the control strain .

Several mechanisms may explain how purA could contribute to acid stress tolerance:

• Energy metabolism: The purA reaction consumes GTP, linking it to energy metabolism. Under acid stress, proper energy management is crucial for maintaining cellular homeostasis, including proton pumping to regulate intracellular pH.

• ATP generation: Studies on recombinant L. plantarum expressing argG showed increased intracellular ATP levels under acid stress . As purA functions in a parallel metabolic pathway, it may similarly influence ATP pools.

• Intracellular pH regulation: Expression of argG in L. plantarum resulted in improved maintenance of intracellular pH under acidic conditions . Similar mechanisms might involve purA.

• Cross-talk with stress response pathways: Purine metabolism enzymes may interact with or influence stress response pathways. In the argG-expressing L. plantarum strain, expression levels of stress response genes (hsp1, cfa) were increased under acid stress .

The potential role of purA in acid tolerance could be investigated by:

• Comparing purA expression levels under normal and acidic conditions

• Creating purA-overexpressing strains and assessing their acid tolerance

• Analyzing metabolic changes in these strains under acid stress

• Determining whether purA influences intracellular pH and ATP levels similar to argG

What are the potential biotechnological applications of recombinant L. plantarum expressing purA?

Recombinant L. plantarum expressing purA offers several promising biotechnological applications, leveraging both the properties of the host organism and the specific enzymatic activity:

• Enhanced Probiotic Properties: L. plantarum is already recognized for its probiotic characteristics . Engineered strains with modified purine metabolism through purA overexpression could potentially exhibit enhanced survival in the gastrointestinal tract, where acidic conditions and bile salts present significant stresses.

• Vaccine Delivery System: L. plantarum has been successfully used as a mucosal vaccine delivery platform . Recombinant L. plantarum expressing both purA (for improved survival) and antigen proteins could serve as an effective oral or intranasal vaccine delivery system. Studies have shown that oral and intranasal immunization with recombinant L. plantarum can induce effective mucosal, cellular, and systemic immune responses .

• Biocatalysis: Purified recombinant purA could be utilized for the enzymatic synthesis of adenylosuccinate and related nucleotide derivatives for pharmaceutical applications.

• Metabolic Engineering: Modulating purA expression could redirect metabolic flux through purine pathways, potentially enhancing the production of valuable nucleotide-derived compounds.

• Cell Factories for Nucleotide Production: Engineered L. plantarum strains with optimized purA expression could serve as cell factories for the production of nucleotides and nucleotide derivatives.

• Food and Dairy Applications: Given L. plantarum's extensive use in food fermentation, recombinant strains with enhanced stress tolerance through purA modification could have applications in challenging food processing environments.

• Research Tool: Recombinant purA with affinity tags can serve as a valuable research tool for studying protein-protein interactions and metabolic networks in lactic acid bacteria.

The development of these applications would require careful optimization of expression systems, as has been demonstrated for other recombinant proteins in L. plantarum , and appropriate regulatory considerations given L. plantarum's status as a food-grade organism.

How can site-directed mutagenesis be used to study the catalytic mechanism of L. plantarum purA?

Site-directed mutagenesis is a powerful approach for investigating the catalytic mechanism of purA from L. plantarum. By systematically altering specific amino acid residues and assessing the effects on enzyme activity, researchers can identify residues critical for substrate binding, catalysis, and structural integrity.

A comprehensive mutagenesis study of L. plantarum purA might target:

• Active Site Residues: Based on sequence alignments with well-characterized adenylosuccinate synthetases from other organisms, key residues in the active site can be identified and mutated. Typical targets include:

  • Residues involved in IMP binding

  • Aspartate binding site residues

  • GTP binding pocket residues

  • Magnesium coordination sites

  • Catalytic residues directly involved in the reaction chemistry

• Conserved Motifs: Adenylosuccinate synthetases contain several highly conserved sequence motifs. Mutations within these motifs can reveal their functional importance:

  • GTP-binding motifs (e.g., P-loop)

  • IMP-binding regions

  • Aspartate-binding residues

• Protein-Protein Interaction Interfaces: If purA functions as a dimer or higher-order oligomer, residues at the oligomerization interface can be mutated to assess the importance of oligomerization for activity.

The effects of mutations can be assessed using:

• Enzyme Kinetics: Determining changes in kinetic parameters (Km, kcat, kcat/Km) for each substrate

• Substrate Binding Studies: Using techniques like isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to measure binding affinities

• Structural Analysis: CD spectroscopy or thermal stability assays to assess effects on protein folding and stability

• Computational Modeling: Molecular dynamics simulations to predict and interpret the effects of mutations

Table 1: Example of a systematic mutagenesis approach for L. plantarum purA

This systematic approach can provide detailed insights into the structure-function relationships of L. plantarum purA and potentially identify residues that could be targeted for protein engineering to enhance catalytic efficiency or substrate specificity.

What are the optimal growth and induction conditions for maximizing recombinant purA yield in L. plantarum?

Maximizing recombinant purA yield in L. plantarum requires careful optimization of growth and induction conditions. Based on studies of recombinant protein expression in L. plantarum, several key parameters should be considered:

• Medium Composition: The nutritional environment significantly impacts recombinant protein production. Key components to optimize include:

  • Carbon sources: Type and concentration of sugars

  • Nitrogen sources: Peptones, yeast extract, amino acids

  • Inorganic salts: Particularly Na₂HPO₄, which has been shown to affect recombinant protein production in L. plantarum

  • Growth factors: Inulin has demonstrated significant effects on protein production in L. plantarum

  • Specific amino acids: Leucine has been identified as important for certain recombinant proteins in L. plantarum

• Growth Parameters:

  • Temperature: Typically 30-37°C during growth phase, potentially lowered to 25-30°C during induction

  • pH: L. plantarum can grow at pH values as low as 3.7 , but optimal protein expression may occur at higher pH

  • Aeration: As L. plantarum is microaerophilic, oxygen levels should be carefully controlled

• Induction Strategy (for inducible systems):

  • Cell density at induction: Typically mid-logarithmic phase

  • Inducer concentration: Optimized to balance protein yield and cellular stress

  • Duration of induction: Longer periods may increase yield but can also lead to protein degradation

A systematic optimization approach using design of experiments (DOE) methodology, particularly Box-Behnken design and response surface methodology, has been successful for optimizing recombinant protein production in L. plantarum . This approach allows for evaluation of multiple parameters simultaneously and identification of interaction effects.

In one study optimizing the production of cell envelope proteinase in L. plantarum LP69, the optimized medium composition resulted in a 49.2% increase in enzyme activity and a 120% increase in specific activity compared to pre-optimization conditions . Similar approaches could be applied to optimize purA expression.

What advanced analytical techniques can be used to study the integration of purA in the metabolic network of L. plantarum?

Understanding how purA integrates into the broader metabolic network of L. plantarum requires sophisticated analytical techniques that can capture the complexity of metabolic interactions:

• Metabolomics:

  • Targeted metabolomics: Quantification of specific metabolites in the purine biosynthesis pathway

  • Untargeted metabolomics: Comprehensive profiling of metabolites to identify unexpected connections

  • Isotope tracing: Using isotopically labeled precursors to track metabolic flux through purA and connected pathways

• Transcriptomics:

  • RNA-seq analysis comparing wild-type and purA-modified strains

  • Time-course studies to capture dynamic changes in gene expression

  • Differential expression analysis under various stress conditions

• Proteomics:

  • Quantitative proteomics to identify changes in protein levels

  • Phosphoproteomics to detect regulatory post-translational modifications

  • Protein-protein interaction studies using affinity purification coupled with mass spectrometry

• Metabolic Flux Analysis:

  • ¹³C-metabolic flux analysis to quantify in vivo reaction rates

  • Flux balance analysis using genome-scale metabolic models

  • Dynamic flux modeling to capture temporal changes in metabolism

• Systems Biology Integration:

  • Multi-omics data integration

  • Network analysis to identify regulatory hubs

  • Machine learning approaches to predict metabolic responses

These techniques can reveal how purA activity influences:

• Energy metabolism (ATP/GTP levels)

• Nucleotide pools and their regulation

• Stress response pathways

• Cell growth and division

• Interactions with other metabolic pathways

How can protein-protein interactions involving purA in L. plantarum be effectively identified and characterized?

Identifying and characterizing protein-protein interactions (PPIs) involving purA can provide crucial insights into its regulation and metabolic integration. Several complementary approaches can be employed:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged purA (His-tag, FLAG-tag, etc.) in L. plantarum

  • Purify purA under conditions that preserve protein-protein interactions

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions using reciprocal pulldowns

• Crosslinking Mass Spectrometry (XL-MS):

  • Treat living L. plantarum cells with protein crosslinkers

  • Isolate and digest crosslinked protein complexes

  • Identify crosslinked peptides by specialized mass spectrometry analysis

  • Map interaction interfaces at amino acid resolution

• Bacterial Two-Hybrid (B2H) Systems:

  • Adapt B2H systems for use in L. plantarum or use heterologous hosts

  • Screen for interactions between purA and candidate proteins

  • Use as a validation method for interactions identified by other techniques

• Bimolecular Fluorescence Complementation (BiFC):

  • Split a fluorescent protein into two non-fluorescent fragments

  • Fuse these fragments to purA and potential interacting partners

  • Reconstitution of fluorescence indicates protein-protein interaction in vivo

  • Allows visualization of interaction locations within the cell

• Proximity-Dependent Biotin Identification (BioID):

  • Fuse purA to a biotin ligase (BirA*)

  • The ligase biotinylates nearby proteins in living cells

  • Purify biotinylated proteins and identify by mass spectrometry

  • Identifies both stable and transient interactions

• Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC):

  • Use purified proteins to quantitatively measure direct interactions

  • Determine binding affinities and kinetics

  • Assess the effects of mutations or ligands on interactions

When analyzing PPIs involving purA, particular attention should be paid to:

• Interactions with other enzymes in the purine biosynthesis pathway

• Potential regulatory proteins (kinases, transcription factors)

• Multienzyme complex formation

• Interactions that change under stress conditions (pH, temperature, nutrient limitation)

The combination of multiple complementary techniques provides the most comprehensive view of the protein interaction network surrounding purA in L. plantarum.