Recombinant Chromobacterium violaceum Chorismate synthase (aroC)

What is Chorismate synthase and what role does it play in Chromobacterium violaceum?

Chorismate synthase (aroC, EC 4.2.3.5) is a critical enzyme in the shikimate pathway that catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate. In Chromobacterium violaceum, as in other bacteria, this enzyme catalyzes the sixth step in the seven-step shikimate pathway, which is essential for the biosynthesis of aromatic compounds . The reaction involves a 1,4-elimination of phosphate and loss of a proton from the C-6 hydrogen. This step is crucial for bacterial growth since chorismate serves as a precursor molecule for the synthesis of aromatic amino acids (phenylalanine, tyrosine, and tryptophan), folate, ubiquinone, and siderophores .

Why is Chromobacterium violaceum Chorismate synthase of interest to researchers?

C. violaceum Chorismate synthase attracts significant research interest for several reasons:

• Antimicrobial target potential: The shikimate pathway is present in bacteria, fungi, plants, and apicomplexan parasites but absent in mammals, making it an attractive target for antimicrobial drug development with potentially minimal side effects .

• Unique catalytic mechanism: The reaction catalyzed by Chorismate synthase is unique in nature and requires reduced FMN as a cofactor, though it is not consumed during the reaction .

• Connection to virulence factors: In C. violaceum, the shikimate pathway connects to the production of violacein and other secondary metabolites that contribute to pathogenicity and antimicrobial properties .

• Model system: C. violaceum serves as a model organism for studying quorum sensing and bacterial social interactions, with aromatic compound production being regulated by these mechanisms .

What expression systems are most effective for producing recombinant C. violaceum Chorismate synthase?

E. coli is the most commonly used expression system for C. violaceum Chorismate synthase, as evidenced by commercial preparations . When expressing aroC from C. violaceum, researchers should consider the following methodological approaches:

• Vector selection: pET expression vectors under the control of T7 promoter have shown good results for aroC expression with suitable affinity tags (His-tag is commonly used).

• Expression conditions: Optimal expression is typically achieved at lower temperatures (16-25°C) after induction, which helps prevent inclusion body formation.

• Codon optimization: Codon optimization for E. coli may improve expression yields, particularly given the different GC content between C. violaceum and E. coli.

• Co-expression strategies: Consider co-expression with chaperones if solubility issues are encountered.

The full-length protein (366 amino acids) can be expressed with the sequence as provided in the product datasheet , though construct optimization may be necessary depending on research goals.

What are the most effective purification strategies for obtaining high-purity recombinant Chorismate synthase?

Purification of C. violaceum Chorismate synthase typically involves:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices is effective for His-tagged constructs.

• Intermediate purification: Ion exchange chromatography (typically anion exchange) can remove contaminating proteins and nucleic acids.

• Polishing step: Size exclusion chromatography helps achieve >95% purity and remove aggregates.

• Buffer considerations:

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol) to maintain enzyme activity

  • Phosphate or Tris buffers (pH 7.0-8.0) are typically used

  • Consider adding 10-15% glycerol for stability

For optimal stability, the purified enzyme should be stored at -80°C in the presence of 20-50% glycerol to prevent repeated freeze-thaw cycles .

What are the established methods for measuring Chorismate synthase activity in vitro?

Chorismate synthase activity can be measured using several complementary approaches:

• Spectrophotometric assays:

  • Direct method: Monitoring the disappearance of EPSP at 275 nm

  • Coupled assay: Monitoring FMN oxidation/reduction at 450 nm

  • Following chorismate formation via downstream products

• HPLC-based assays:

  • Separation and quantification of substrate and product

  • Can be coupled with mass spectrometry for enhanced sensitivity

• Continuous vs. discontinuous assays:

  • Continuous assays allow real-time monitoring but may suffer from interference

  • Discontinuous assays involve sampling at time intervals followed by product analysis

The reaction requires reduced FMN (which is not consumed), and activity measurements should include controls for spontaneous decomposition of EPSP and verify the FMN dependency .

How can researchers optimize assay conditions for C. violaceum Chorismate synthase to obtain reliable kinetic parameters?

To optimize assay conditions:

• Buffer selection:

  • pH optimization (typically 7.0-7.5)

  • Ionic strength optimization (50-100 mM buffer is common)

  • Addition of metal ions (investigating potential activators)

• Temperature effects:

  • Temperature optima determination (typically 25-37°C)

  • Thermal stability studies to ensure enzyme stability during assay

• Substrate concentration range:

  • Determine Km for EPSP (typically in the μM range)

  • Ensure substrate concentrations span 0.2-5× Km

• FMN reduction system:

  • Test different reducing systems (dithionite, NADPH/flavodoxin/flavodoxin reductase)

  • Optimize FMN concentration

• Data analysis considerations:

  • Use appropriate enzyme kinetic models

  • Account for potential substrate inhibition at high concentrations

  • Consider allosteric effects

These optimizations will enable reliable determination of kinetic parameters such as Km, kcat, and potential inhibition constants .

What is known about the structural features of C. violaceum Chorismate synthase and how do they relate to its mechanism?

While a detailed crystal structure specific to C. violaceum Chorismate synthase is not available in the provided search results, comparative analysis with other bacterial Chorismate synthases suggests:

• Key structural features:

  • The enzyme likely adopts a core αβα sandwich fold

  • The active site contains a conserved FMN binding pocket

  • Critical residues include those involved in EPSP binding and phosphate elimination

• Mechanistic implications:

  • The unique reaction requires reduced FMN but does not consume it

  • The proposed mechanism involves electron transfer from FMN to phosphate, facilitating its elimination

  • The substrate may temporarily donate an electron for FMN regeneration

• Conserved regions:

  • FMN-binding domain

  • Substrate recognition elements

  • Catalytic residues involved in proton abstraction

Understanding these structural elements is essential for rational design of inhibitors that could serve as antimicrobial agents .

How does the proposed catalytic mechanism of C. violaceum Chorismate synthase differ from other enzymes in the shikimate pathway?

The catalytic mechanism of Chorismate synthase stands out in the shikimate pathway due to several unique features:

• Cofactor requirements:

  • Requires reduced FMN as an electron donor

  • Unlike other oxidoreductases, the FMN is not oxidized during the reaction

  • The FMN likely participates in single-electron transfer reactions

• Chemical transformation:

  • Catalyzes an anti-1,4-elimination reaction (phosphate elimination and proton abstraction)

  • Creates a second double bond in the ring structure

  • Does not involve redox change in the substrate

• Proposed key steps:

  • Initial binding of EPSP in proximity to reduced FMN

  • Single electron transfer from FMN to phosphate group

  • Phosphate elimination creating a radical intermediate

  • Proton abstraction from C-6 position

  • Electron return to FMN and formation of chorismate

This mechanism differs significantly from other shikimate pathway enzymes, which typically catalyze more conventional chemical transformations without requiring such specialized cofactor interactions .

What approaches have been successful in developing inhibitors of Chorismate synthase for antimicrobial applications?

Research on Chorismate synthase inhibitors has explored several promising strategies:

• Substrate analogs:

  • EPSP-like compounds with modifications at the phosphate position

  • Compounds mimicking the transition state of the reaction

  • Stable analogs of reactive intermediates

• Cofactor-based inhibitors:

  • Compounds that compete with FMN binding

  • Modified flavins that disrupt the electron transfer process

• Allosteric inhibitors:

  • Compounds binding outside the active site to alter enzyme conformation

  • Molecules disrupting protein-protein interactions in multimeric forms

• Fragment-based approaches:

  • Building inhibitors by combining small fragments with affinity for different enzyme regions

  • Structure-guided optimization of initial fragment hits

The unique nature of the reaction and the absence of the shikimate pathway in mammals make these inhibitors promising candidates for broad-spectrum antimicrobials with potentially minimal side effects .

How can researchers assess the specificity and potency of inhibitors against C. violaceum Chorismate synthase?

To evaluate inhibitor specificity and potency:

• In vitro enzyme assays:

  • Determination of IC50 values using standardized assay conditions

  • Kinetic analysis to determine inhibition type (competitive, non-competitive, uncompetitive)

  • Structure-activity relationship studies of related compounds

• Selectivity profiling:

  • Testing against human enzymes to assess potential off-target effects

  • Comparison with effects on other shikimate pathway enzymes

  • Cross-species comparison with Chorismate synthases from different organisms

• Cellular assays:

  • Growth inhibition assays against C. violaceum and other bacteria

  • Verification that growth inhibition correlates with enzyme inhibition

  • Assessment of resistance development potential

• Computational approaches:

  • Molecular docking studies to predict binding modes

  • Molecular dynamics simulations to understand inhibitor-enzyme interactions

  • Virtual screening to identify new inhibitor candidates

These approaches provide a comprehensive assessment of inhibitor properties and help guide optimization efforts .

How is Chorismate synthase regulated in C. violaceum and how does this affect bacterial physiology?

Regulation of Chorismate synthase in C. violaceum involves multiple layers of control:

• Transcriptional regulation:

  • Potential feedback inhibition by pathway end products (aromatic amino acids)

  • Quorum sensing may influence expression under certain conditions

  • Response to environmental stresses like nutrient limitation

• Post-translational regulation:

  • Allosteric regulation by metabolites

  • Potential protein-protein interactions

  • Availability of the FMN cofactor

• Pathway integration:

  • Coordination with other shikimate pathway enzymes

  • Balance with branching pathways from chorismate

  • Connection to secondary metabolite production

Research has shown that disruption of quorum sensing through enzymatic degradation of acyl-homoserine lactones (AHLs) affects the production of violacein and other secondary metabolites in C. violaceum, suggesting a potential regulatory connection between quorum sensing and aromatic compound biosynthesis pathways .

What is the relationship between Chorismate synthase activity and the production of virulence factors in C. violaceum?

The connection between Chorismate synthase and virulence factors in C. violaceum is multifaceted:

• Violacein production:

  • Chorismate serves as a precursor for tryptophan, which is further processed to produce violacein

  • Violacein has antimicrobial properties that may provide competitive advantages

  • Quorum sensing regulation links population density to violacein production

• Other secondary metabolites:

  • Chorismate is a branch point for multiple secondary metabolite pathways

  • C. violaceum produces other antibiotics including anisomycin, aerocyanidin, and aerocavin

  • These compounds may contribute to pathogenicity and ecological competitiveness

• Iron acquisition:

  • Chorismate is a precursor for siderophores that facilitate iron uptake

  • Iron acquisition is critical for virulence in many pathogens

  • Inhibition of Chorismate synthase would affect siderophore production

• Integrated response systems:

  • Stress responses may coordinate chorismate production with virulence factor expression

  • The antibiotic-induced response (air) two-component regulatory system connects antibiotic sensing to violacein production

Understanding these connections provides insight into how targeting Chorismate synthase might affect bacterial virulence beyond simple growth inhibition .

How does C. violaceum Chorismate synthase compare structurally and functionally with the enzyme from other bacterial species?

Comparative analysis of Chorismate synthases across bacterial species reveals important similarities and differences:

• Structural conservation:

  • Core catalytic domains are generally conserved across bacterial species

  • FMN binding sites show high sequence conservation

  • Species-specific variations occur primarily in peripheral regions

• Functional differences:

  • Kinetic parameters (Km, kcat) vary between species

  • Sensitivity to inhibitors can differ significantly

  • Cofactor requirements (specifically for FMN reduction) may vary

• Bifunctional vs. monofunctional forms:

  • Bacterial forms (including C. violaceum) are typically monofunctional

  • Fungal forms often have a fused NADPH-dependent flavin reductase domain

  • This evolutionary difference has implications for inhibitor design

• Oligomeric state:

  • Bacterial Chorismate synthases typically function as tetramers

  • Quaternary structure can influence catalytic efficiency and regulation

These comparisons help identify conserved features that might be targeted by broad-spectrum inhibitors versus species-specific features that could enable selective targeting .

What insights can be gained from studying the evolution of Chorismate synthase across different bacterial lineages?

Evolutionary analysis of Chorismate synthase offers several valuable insights:

• Phylogenetic relationships:

  • Sequence conservation reflects taxonomic relationships

  • Horizontal gene transfer events may be identified through incongruent phylogenies

  • Evolutionary rate analysis can identify regions under selective pressure

• Adaptation to ecological niches:

  • Variations in enzyme properties may reflect adaptation to different environments

  • C. violaceum's enzyme may have specific adaptations to its soil/water habitat

  • Pathogenic vs. non-pathogenic strains may show characteristic differences

• Coevolution with other pathway components:

  • Coordinated evolution with other shikimate pathway enzymes

  • Coevolution with regulatory elements

  • Adaptation to different end-product profiles

• Implications for inhibitor design:

  • Highly conserved regions represent potential broad-spectrum targets

  • Variable regions may enable species-specific targeting

  • Understanding evolutionary constraints helps predict resistance development

This evolutionary perspective provides context for understanding enzyme function and informs more sophisticated approaches to inhibitor design .

How can C. violaceum Chorismate synthase be utilized in multi-enzyme cascade reactions for biotechnological applications?

C. violaceum Chorismate synthase can be integrated into multi-enzyme cascades for various biotechnological purposes:

• Biocatalytic production of aromatic compounds:

  • One-pot enzymatic synthesis of chorismate-derived products

  • Combined with downstream enzymes to produce specialty chemicals

  • Engineered to accept non-natural substrates for novel compound synthesis

• Metabolic engineering applications:

  • Reconstitution of partial or complete shikimate pathway in vitro

  • Optimization of flux through chorismate for overproduction of valuable metabolites

  • Bypassing regulatory constraints present in vivo

• Biosensor development:

  • Coupling chorismate synthase activity to detection systems

  • Monitoring shikimate pathway activity in complex biological samples

  • Detecting inhibitors or pathway intermediates

• Methodological considerations:

  • Enzyme immobilization strategies to enhance stability and reusability

  • Cofactor regeneration systems for FMN reduction

  • Compartmentalization approaches to overcome incompatible reaction conditions

These applications leverage the unique catalytic capabilities of Chorismate synthase within controlled environments designed to maximize productivity and efficiency .

What are the most promising approaches for engineering C. violaceum Chorismate synthase to improve its catalytic properties?

Advanced protein engineering of C. violaceum Chorismate synthase could focus on:

• Rational design strategies:

  • Structure-guided mutations of active site residues

  • Engineering cofactor binding for enhanced affinity or altered specificity

  • Stabilizing mutations to improve thermostability or solvent tolerance

• Directed evolution approaches:

  • Error-prone PCR to generate variant libraries

  • DNA shuffling with Chorismate synthases from other species

  • Selection or screening systems to identify improved variants

• Specific improvements to target:

  • Increased catalytic efficiency (kcat/Km)

  • Reduced product inhibition

  • Broader substrate scope for non-natural substrates

  • Enhanced stability under industrial conditions

• Computational approaches:

  • Molecular dynamics simulations to identify flexible regions

  • In silico screening of mutant libraries

  • Machine learning prediction of beneficial mutations

• Experimental validation methods:

  • High-throughput activity assays

  • Structural characterization of engineered variants

  • In vivo testing in model systems

These engineering approaches could yield enzymes with enhanced properties for both fundamental research and biotechnological applications, including improved biocatalysts for the production of aromatic compounds .

What are the common technical challenges in working with recombinant C. violaceum Chorismate synthase and how can they be overcome?

Researchers frequently encounter these challenges when working with recombinant C. violaceum Chorismate synthase:

• Solubility issues:

  • Solution: Use solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Solution: Optimize expression conditions (lower temperature, reduced inducer concentration)

  • Solution: Add solubility enhancers to lysis/purification buffers (glycerol, arginine, low concentrations of mild detergents)

• Cofactor binding and activity:

  • Solution: Ensure complete reduction of FMN using appropriate reducing systems

  • Solution: Optimize FMN:protein ratio in assays

  • Solution: Use anaerobic conditions to prevent FMN oxidation

• Substrate stability:

  • Solution: Prepare EPSP fresh or store properly to prevent decomposition

  • Solution: Include controls for spontaneous substrate degradation

  • Solution: Consider enzymatic synthesis of EPSP immediately before use

• Assay interference:

  • Solution: Account for background absorbance of FMN in spectrophotometric assays

  • Solution: Use HPLC or mass spectrometry for more specific product detection

  • Solution: Include appropriate controls for non-enzymatic reactions

• Protein stability during storage:

  • Solution: Store at -80°C with 20-50% glycerol

  • Solution: Avoid repeated freeze-thaw cycles

  • Solution: Test addition of reducing agents and protease inhibitors

Addressing these challenges systematically will improve experimental outcomes and data reliability .

How can researchers effectively integrate multi-omics approaches to study the role of Chorismate synthase in C. violaceum metabolism?

Multi-omics integration for studying Chorismate synthase in C. violaceum requires systematic approaches:

• Experimental design considerations:

  • Coordinated sampling for different omics analyses

  • Inclusion of appropriate controls (e.g., enzyme inhibition, gene knockout)

  • Time-course studies to capture dynamic responses

• Integration of complementary techniques:

  • Genomics: Identify gene clusters and potential regulatory elements

  • Transcriptomics: Measure expression changes under various conditions

  • Proteomics: Quantify protein levels and post-translational modifications

  • Metabolomics: Track metabolite fluxes through the shikimate pathway

  • Fluxomics: Measure carbon flow using labeled precursors

• Data analysis approaches:

  • Statistical methods for integrating heterogeneous data types

  • Pathway analysis to contextualize findings

  • Mathematical modeling of metabolic networks

• Case study example:

  • Research has demonstrated the power of combined metabolomic and proteomic approaches to study the impact of quorum sensing disruption on C. violaceum metabolism

  • Regularized Canonical Correlation Analysis (rCCA) effectively linked proteomic and metabolomic data to identify relationships between proteins and metabolites

  • This approach revealed significant correlations between quorum sensing, violacein production, and other metabolic pathways

These integrated approaches provide a more comprehensive view of how Chorismate synthase functions within the broader metabolic network of C. violaceum .

What emerging technologies might advance our understanding of C. violaceum Chorismate synthase function and regulation?

Several cutting-edge technologies show promise for deepening our understanding of Chorismate synthase:

• Advanced structural biology techniques:

  • Cryo-electron microscopy for visualization of enzyme-substrate complexes

  • Time-resolved crystallography to capture reaction intermediates

  • Neutron diffraction to precisely locate hydrogen atoms in the active site

• Single-molecule approaches:

  • Single-molecule FRET to monitor conformational changes during catalysis

  • Optical tweezers or atomic force microscopy to study enzyme mechanics

  • Single-enzyme kinetics to reveal heterogeneity in catalytic behavior

• Advanced computational methods:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations of the reaction mechanism

  • Machine learning for prediction of enzyme-substrate interactions

  • Network analysis of metabolic pathways centered on chorismate

• Genome editing technologies:

  • CRISPR-Cas9 for precise genome editing in C. violaceum

  • Base editing for introducing point mutations without double-strand breaks

  • CRISPRi/CRISPRa for reversible modulation of gene expression

• Systems biology approaches:

  • Whole-cell modeling incorporating Chorismate synthase within metabolic networks

  • Synthetic biology redesign of shikimate pathway regulation

  • Integration of multi-omics data with artificial intelligence for pathway prediction

These technologies promise to reveal new insights into Chorismate synthase function and regulation at unprecedented resolution .

What are the most significant unanswered questions regarding C. violaceum Chorismate synthase that warrant further investigation?

Several critical knowledge gaps remain regarding C. violaceum Chorismate synthase:

Addressing these questions would significantly advance our understanding of this enzyme and potentially lead to new antimicrobial strategies and biotechnological applications .