Recombinant Photobacterium profundum Chorismate synthase (aroC)

What is the role of chorismate synthase in the shikimate pathway?

Chorismate synthase (AroC) catalyzes the seventh and final step in the shikimate pathway, converting 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate. This reaction is particularly important as chorismate represents the branch point for the biosynthesis of aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and other aromatic compounds. The shikimate pathway synthesizes chorismate through seven sequential enzymatic steps starting from erythrose-4-phosphate and phosphoenolpyruvate .

Importantly, this pathway cannot be bypassed through any alternative enzymatic route, making chorismate synthase an essential enzyme for organisms that possess the shikimate pathway. Inhibition of this pathway can lead to cell death in these organisms, which includes bacteria, fungi, and plants . The absence of this pathway in mammals makes chorismate synthase a promising target for the development of antibiotics, herbicides, and fungicides.

Why is Photobacterium profundum an interesting organism for studying chorismate synthase?

Photobacterium profundum is a psychrohalophilic bacterium isolated from deep-sea sediments, capable of thriving in cold, high-pressure, and high-salinity environments . Enzymes from extremophiles like P. profundum often possess unique structural and functional adaptations that allow them to function optimally under extreme conditions.

These adaptations may include:

• Structural modifications that maintain flexibility at low temperatures

• Alterations in amino acid composition that provide stability under high pressure

• Unique salt bridges or other ionic interactions that contribute to halophilic properties

While specific information about P. profundum chorismate synthase is limited in the current literature, studies of other enzymes from this organism, such as α-carbonic anhydrase (PprCA), reveal significant adaptations to extreme environments . PprCA maintains catalytic activity under conditions that would denature enzymes from mesophilic organisms, suggesting that P. profundum chorismate synthase might similarly exhibit exceptional stability and activity profiles under extreme conditions.

How does the shikimate pathway differ between prokaryotes and eukaryotes?

The shikimate pathway is present in prokaryotes (bacteria), lower eukaryotes (fungi), and plants, but is notably absent in mammals . This taxonomic distribution makes it an excellent target for developing selective antimicrobial and herbicidal compounds.

Key differences between prokaryotic and eukaryotic shikimate pathways include:

In prokaryotes like Photobacterium profundum, the shikimate pathway enzymes are often subject to regulation through protein-protein interactions. This is evidenced by studies of related enzymes such as chorismate mutase from Mycobacterium tuberculosis (MtCM), which requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity .

What are the key considerations for cloning and expressing recombinant P. profundum chorismate synthase?

When designing experiments for cloning and expressing recombinant P. profundum chorismate synthase, researchers should consider several factors specific to this psychrohalophilic organism:

• Codon optimization: P. profundum's codon usage may differ from common expression hosts like E. coli. Consider synthesizing a codon-optimized gene for improved expression.

• Expression system selection: Based on protocols used for other P. profundum proteins, an approach similar to that used for α-carbonic anhydrase might be effective:

  • Gene amplification using PCR with gene-specific primers

  • Cloning into appropriate expression vectors (pET systems are commonly used)

  • Expression in a bacterial host system like E. coli BL21(DE3)

• Expression conditions: Due to P. profundum's psychrohalophilic nature, protein folding may be optimized at lower temperatures. Consider induction at 16-20°C rather than the standard 37°C to improve solubility and proper folding.

• Purification strategy: A multi-step purification process is recommended:

  • Initial capture using affinity chromatography (His-tag purification is common)

  • Further purification using size exclusion chromatography to separate monomeric and potential oligomeric forms

  • Activity verification using enzyme-specific assays

• Buffer composition: Include stabilizing agents appropriate for psychrohalophilic enzymes, such as glycerol or specific salt concentrations that mimic the native environment.

How can researchers optimize enzyme activity assays for P. profundum chorismate synthase?

Optimizing enzyme activity assays for P. profundum chorismate synthase requires consideration of its psychrohalophilic origins:

• Temperature and pressure conditions: Unlike mesophilic enzymes, P. profundum enzymes may show optimal activity at lower temperatures (4-15°C) and potentially under increased pressure. Design assays with temperature control capabilities, and if possible, equipment that can simulate high-pressure environments.

• Buffer composition: Test multiple buffer systems with varying salt concentrations (particularly NaCl) to determine optimal conditions for enzyme activity. Based on studies of other P. profundum enzymes, higher salt concentrations may be necessary for optimal activity .

• pH optimization: Conduct initial experiments across a range of pH values (typically pH 6-9) to determine the optimal pH for activity. Psychrohalophilic enzymes sometimes exhibit pH optima different from their mesophilic counterparts.

• Substrate concentration optimization: Determine appropriate substrate concentration ranges by performing preliminary kinetic experiments. For chorismate synthase, EPSP would be the substrate of interest.

• Experimental design considerations: As noted in general experimental design principles, randomization is important, but with small sample sizes, it may not produce exactly equivalent groups . Statistical control for known differences should be implemented, and researchers should be aware that differences observed may reflect inherent experimental variation rather than treatment effects.

What structural characteristics might be expected in P. profundum chorismate synthase?

While specific structural information about P. profundum chorismate synthase is not available in the current literature, insights can be drawn from studies of other P. profundum enzymes and chorismate synthases from different organisms:

• Core structural conservation: The catalytic core of chorismate synthase is likely to be conserved across species, maintaining essential residues for substrate binding and catalysis.

• Cold adaptation features: Based on the crystal structure of P. profundum α-carbonic anhydrase (PprCA), several features associated with cold adaptation might be expected:

  • Reduced number of surface loops compared to mesophilic homologs

  • Unique dimer interfaces that may contribute to stability under extreme conditions

  • Possible presence of specific ion binding sites that stabilize the structure

• Quaternary structure: P. profundum α-carbonic anhydrase exists as a heterogeneous mixture of monomers and dimers . Chorismate synthase from P. profundum might similarly exhibit multiple oligomeric states that could be functionally relevant.

• Active site architecture: The active site is likely to contain conserved catalytic residues while potentially featuring adaptations that maintain flexibility and activity at low temperatures.

• Surface properties: The protein surface may show an increased proportion of charged residues, which is a common adaptation in halophilic proteins to maintain solubility in high-salt environments.

How do directed evolution approaches help understand enzyme function as demonstrated in related systems?

Directed evolution offers powerful insights into enzyme function and potential for optimization, as demonstrated in studies of related enzymes:

• Uncovering evolutionary potential: Research on Mycobacterium tuberculosis chorismate mutase (MtCM) revealed that an enzyme that naturally evolved for "mediocrity" (having less than 1% of the catalytic efficiency of typical chorismate mutases) could be dramatically improved through directed evolution .

• Methodological approach: Successful directed evolution typically involves:

  • Iterative cycles of mutagenesis and selection

  • Screening for improved properties (catalytic efficiency, stability, etc.)

  • Structural analysis of evolved variants to understand the molecular basis of improvements

• Magnitude of improvement: In the case of MtCM, directed evolution raised the catalytic efficiency (kcat/Km) 270-fold, from a naturally compromised state to a highly efficient enzyme with activity comparable to or exceeding that of naturally efficient enzymes .

• Structural basis of improvements: Crystal structures of evolved variants revealed that some acquired mutations were in conserved positions found in naturally efficient enzymes, while others were in unexpected positions beyond the active site .

• Evolutionary insights: Such studies reveal that naturally "mediocre" enzymes often have inherent potential for much higher activity, suggesting that natural selection sometimes favors regulated mediocrity to enable allosteric control and pathway regulation . This perspective might be relevant for understanding P. profundum chorismate synthase and its evolutionary adaptations.

What approaches are being used to develop inhibitors of chorismate synthase?

Developing inhibitors for chorismate synthase is an active area of research due to its potential as an antimicrobial target. Recent research approaches include:

• Virtual screening and molecular dynamics: Computational approaches have successfully identified potential inhibitor scaffolds, as demonstrated in work on Paracoccidioides brasiliensis chorismate synthase .

• Structure-based design: Using crystallographic data to guide rational design of inhibitors that interact with key active site residues.

• SAR development: Establishing structure-activity relationships by synthesizing series of related compounds with systematic modifications, as seen in the development of naphthalene-based inhibitors with Kd values up to 19 μM for P. brasiliensis chorismate synthase .

• Iterative optimization: Moving from first-generation to second-generation inhibitors through systematic improvements based on binding and inhibition data .

• Comprehensive biological evaluation: Testing candidates through multiple experimental approaches:

  • In vitro enzyme inhibition assays

  • Binding studies (e.g., isothermal titration calorimetry, surface plasmon resonance)

  • Crystallization of enzyme-inhibitor complexes

  • Cytotoxicity studies

These approaches could be adapted for P. profundum chorismate synthase, with particular attention to the enzyme's potential structural adaptations for cold and high-pressure environments.

Why is the shikimate pathway considered a promising antimicrobial target?

The shikimate pathway presents several advantages as an antimicrobial target:

• Taxonomic exclusivity: The pathway is present in bacteria, fungi, and plants but absent in mammals, allowing for selective targeting without directly affecting host metabolic processes .

• Essential function: The pathway produces chorismate, a precursor for aromatic amino acids and various other essential compounds. Inhibition can lead to cell death in organisms that rely on this pathway .

• Non-bypassing nature: The production of chorismate through the seven enzymatic steps of the shikimate pathway "cannot be bypassed through any other enzyme," making it a metabolic bottleneck .

• Proven target: The commercial success of glyphosate, which inhibits 5-enolpyruvylshikimate-3-phosphate synthase (the sixth enzyme in the pathway), demonstrates the viability of targeting this pathway.

• Multiple targetable enzymes: Each of the seven enzymes in the pathway represents a potential target, with chorismate synthase being particularly attractive as the final step in the pathway.

These characteristics make chorismate synthase from P. profundum an interesting subject for antimicrobial development, potentially offering insights into inhibitors that could be effective under extreme environmental conditions where conventional antibiotics might be less effective.

What are common challenges in working with recombinant enzymes from psychrophilic organisms?

Researchers working with recombinant enzymes from psychrophilic organisms like P. profundum often encounter specific challenges:

• Temperature sensitivity: Psychrophilic enzymes often show reduced stability at room or higher temperatures, necessitating careful temperature control during purification and storage.

• Solubility issues: Expression in mesophilic hosts like E. coli can lead to improper folding or aggregation due to temperature mismatch during expression.

• Activity measurement: Standard enzyme assays often performed at room temperature may not accurately reflect the optimal activity of psychrophilic enzymes.

• Structural flexibility: Psychrophilic enzymes typically exhibit greater flexibility to function at low temperatures, which can make them more susceptible to proteolysis during purification.

• Buffer requirements: Psychrohalophilic enzymes like those from P. profundum may require specific ionic conditions to maintain stability and activity, necessitating careful optimization of buffer compositions.

• Heterogeneous oligomeric states: As observed with P. profundum α-carbonic anhydrase, psychrophilic enzymes may exist in multiple oligomeric forms (monomers and dimers), complicating purification and activity analysis .

How can researchers address protein stability issues when working with P. profundum enzymes?

Based on studies of other P. profundum enzymes and general principles for working with psychrophilic proteins, several strategies can help address stability issues:

• Modified expression protocols:

  • Lower induction temperatures (16-20°C)

  • Extended expression times

  • Co-expression with cold-adapted chaperones

• Optimized purification conditions:

  • Conducting all purification steps at 4°C

  • Including protease inhibitors to prevent degradation

  • Using stabilizing additives like glycerol (10-20%)

  • Including specific ions that might stabilize the structure, such as the chloride ion observed in the dimer interface of P. profundum α-carbonic anhydrase

• Storage considerations:

  • Flash-freezing in liquid nitrogen rather than slow freezing

  • Adding cryoprotectants to prevent freeze-thaw damage

  • Aliquoting to avoid repeated freeze-thaw cycles

• Activity preservation:

  • Optimizing buffer conditions (pH, salt concentration)

  • Including stabilizing cofactors or substrate analogs

  • Considering immobilization approaches for long-term stability

• Structural analysis precautions:

  • Modified crystallization conditions, potentially at lower temperatures

  • Careful handling during sample preparation for structural studies

These approaches can help maintain the native properties of P. profundum enzymes during experimental procedures, ensuring that observations reflect their true biochemical and biophysical characteristics rather than artifacts of experimental manipulation.