Recombinant Acinetobacter sp. Chorismate synthase (aroC)

What is the biological role of chorismate synthase in Acinetobacter species?

Chorismate synthase (CS) in Acinetobacter species catalyzes the conversion of 5-enoylpyruvyl-shikimate-3-phosphate (EPSP) to chorismate through the removal of the 3-phosphate group . This reaction represents the final step in the seven-step shikimate pathway, which is critical for the synthesis of folate cofactors, bacterial siderophores, and all aromatic amino acids in bacteria . Chorismate serves as a vital precursor for numerous aromatic compounds essential for bacterial survival and virulence . The enzyme's catalytic function relies on a specific lyase activity that is distinctive from other metabolic enzymes, making it particularly attractive as a potential antimicrobial target .

Why is aroC considered an essential gene in Acinetobacter baumannii?

The aroC gene has been experimentally validated as essential for the in vivo growth and survival of A. baumannii through gene deletion studies . When the aroC gene is deleted, A. baumannii exhibits complete growth inhibition in in vivo infection models, confirming its essentiality . Unlike some other metabolic pathways, the products of the chorismate pathway cannot be supplemented from host tissues during infection, creating an absolute requirement for a functional aroC gene . This essentiality has been directly demonstrated through survival studies in rat soft-tissue infection models, where mutants lacking aroC failed to establish infection .

How does the chorismate pathway differ between bacteria and humans?

The chorismate biosynthetic pathway is completely absent in humans and other mammals, who obtain aromatic amino acids exclusively through dietary sources . This fundamental difference creates a significant selective advantage for targeting this pathway with antimicrobials. The bacterial-specific nature of chorismate synthase means that inhibitors targeting this enzyme should theoretically have minimal off-target effects on human metabolism . This absence in human cells combined with the essentiality of aroC in bacterial survival makes it a particularly appealing target for narrow-spectrum antibiotic development with potentially reduced side effects.

What are the key structural features of Acinetobacter chorismate synthase?

Acinetobacter chorismate synthase is a protein of approximately 43 kDa that belongs to the lyase family of enzymes . While the exact three-dimensional structure of A. baumannii CS has not been fully determined, comparative structural biology approaches have identified critical domains including the active site architecture and substrate binding pockets . The enzyme contains flexible shikimate-binding domains and LID regions that undergo conformational changes during catalysis . These structural elements vary across bacterial species and represent potential targets for species-specific inhibitor design .

What expression systems are optimal for producing recombinant A. baumannii chorismate synthase?

The most effective expression system documented for A. baumannii chorismate synthase utilizes the pET28c vector in Escherichia coli BL21(DE3) cells . This system allows for high-yield expression with a His-tag for simplified purification processes. Optimal expression conditions typically involve induction with IPTG at lower temperatures (16-25°C) to enhance protein solubility . Similar approaches have been successfully employed for expressing chorismate synthase from related bacterial species such as Moraxella catarrhalis, suggesting this system's broad applicability .

How can the enzymatic activity of recombinant chorismate synthase be accurately measured?

Enzymatic activity of recombinant chorismate synthase can be measured through several complementary approaches:

• Spectrophotometric assays: Monitoring the conversion of EPSP to chorismate by tracking changes in absorption at 275 nm, which corresponds to the formation of the conjugated diene structure in chorismate .

• Coupled enzyme assays: Using auxiliary enzymes that react with chorismate to produce detectable products, allowing for real-time measurement of chorismate synthase activity .

• HPLC-based methods: Separating and quantifying substrate and product concentrations directly through chromatographic techniques, offering higher specificity but lower throughput .

For accurate kinetic characterization, assays should be conducted under optimal buffer conditions (typically pH 7.0-7.5) with controlled concentrations of FMN cofactor, which is essential for catalytic activity .

What strategies are most effective for heterologous expression and purification of recombinant A. baumannii chorismate synthase?

For optimal heterologous expression and purification of A. baumannii chorismate synthase, researchers should consider:

• Vector selection: The pET28c vector containing an N-terminal His-tag has proven successful for high-yield expression .

• Host strain optimization: E. coli BL21(DE3) cells are preferred due to their reduced protease activity and efficient protein expression machinery .

• Induction conditions: Lower temperatures (16-20°C) during induction minimize inclusion body formation and enhance soluble protein yield .

• Purification protocol: A two-step purification approach involving initial Ni-NTA affinity chromatography followed by size-exclusion chromatography achieves high purity (>95%) while maintaining enzymatic activity .

• Buffer composition: Including reducing agents (2-5 mM DTT or β-mercaptoethanol) and glycerol (10%) in purification buffers significantly improves protein stability during purification and storage .

This approach typically yields 15-20 mg of purified protein per liter of bacterial culture with retained enzymatic activity .

How can one design knockout experiments to validate the essentiality of aroC in Acinetobacter species?

Designing knockout experiments to validate aroC essentiality requires:

• Gene deletion strategy: Construction of a clean deletion mutant (ΔaroC) using allelic exchange techniques with suicide vectors carrying homologous flanking regions of the aroC gene .

• Complementation controls: Development of a complementation plasmid expressing functional aroC to confirm that observed phenotypes are specifically due to aroC deletion .

• In vitro growth assessment: Comparing growth curves of wild-type, ΔaroC mutant, and complemented strains in both rich and minimal media to distinguish between absolute essentiality versus conditional growth defects .

• In vivo infection models: Using established animal models such as the rat soft tissue infection model to assess bacterial survival and growth over 48-hour periods post-infection . The wild-type strain typically shows approximately 2-log growth over 48 hours, while aroC deletion mutants show complete clearance over the same period, demonstrating in vivo essentiality .

• Metabolite supplementation tests: Determining whether growth defects can be rescued by supplementation with aromatic amino acids or other downstream metabolites of the chorismate pathway .

This comprehensive approach establishes both in vitro and in vivo essentiality profiles, providing foundational evidence for aroC as an antimicrobial target .

What high-throughput screening approaches are most suitable for identifying inhibitors of A. baumannii chorismate synthase?

Effective high-throughput screening approaches for identifying A. baumannii chorismate synthase inhibitors include:

• Virtual screening: Utilizing refined homology models of A. baumannii chorismate synthase for ligand-based virtual screening against compound databases like the Supernatural Database . This approach has successfully identified promising phytochemical inhibitors including caffeic acid, gallic acid, and o-coumaric acid .

• Fluorescence-based activity assays: Developing assays that detect changes in intrinsic fluorescence or use fluorescent reporter systems to monitor enzymatic activity in real-time across multi-well plate formats .

• Differential scanning fluorimetry (DSF): Screening compounds based on their ability to alter the thermal stability of the enzyme, which often correlates with binding affinity .

• Binding validation using isothermal titration calorimetry (ITC): Confirming binding interactions and determining thermodynamic parameters (ΔH, ΔS, Kd) of promising hit compounds, as demonstrated with gallic acid binding to chorismate synthase .

These approaches can be combined in a tiered screening strategy, starting with computational methods to identify promising candidates, followed by in vitro assays to confirm activity and binding properties .

How do the structural and functional properties of chorismate synthase differ across Acinetobacter species and other bacterial pathogens?

Chorismate synthase demonstrates notable structural and functional differences across bacterial species despite catalyzing the same reaction:

• Sequence divergence: While maintaining conserved catalytic residues, chorismate synthases from different bacteria show significant sequence variation in non-catalytic regions, particularly in substrate binding loops and regulatory domains .

• Cofactor requirements: All bacterial chorismate synthases require reduced FMN as a cofactor, but the mechanisms of FMN reduction and binding affinities can vary considerably between species .

• Oligomeric state: Chorismate synthases exist as tetramers in many bacteria, but the inter-subunit interactions and stabilizing forces differ across species, affecting inhibitor binding sites at subunit interfaces .

• Catalytic efficiency: Kinetic parameters vary significantly, with the Km for EPSP ranging from approximately 10-50 μM across different bacterial species, potentially reflecting adaptations to species-specific metabolic requirements .

These differences provide opportunities for developing species-selective inhibitors that target unique structural features of Acinetobacter chorismate synthase while minimizing effects on beneficial microbiota .

What is the relationship between aroC function and antimicrobial resistance mechanisms in Acinetobacter baumannii?

The relationship between aroC function and antimicrobial resistance mechanisms in A. baumannii involves several interconnected aspects:

• Metabolic resilience: Functional chorismate biosynthesis provides metabolic flexibility that can indirectly support resistance mechanisms by maintaining bacterial fitness during antibiotic stress .

• Biofilm formation: Chorismate-derived molecules contribute to biofilm development in A. baumannii, with biofilms being a key physical resistance mechanism that reduces antibiotic penetration and effectiveness .

• Genomic context: While aroC itself is not typically found on mobile genetic elements associated with antimicrobial resistance (e.g., AbaR resistance islands or plasmids), its products support bacterial physiology during stress responses induced by antibiotics .

• Resistance to existing targeting strategies: Unlike enzymes targeted by sulfonamides (folate pathway), no clinically approved antibiotics currently target chorismate synthase, potentially making it effective against multi-drug resistant (MDR) and extensively drug-resistant (XDR) A. baumannii strains .

• Complementary targeting: Combined inhibition of chorismate synthase with existing antibiotics may reduce the likelihood of resistance development, similar to the synergistic approach used with trimethoprim-sulfamethoxazole targeting sequential steps in folate biosynthesis .

This complex relationship suggests that targeting aroC could provide advantages against resistant A. baumannii strains by exploiting metabolic vulnerabilities distinct from current antibiotic targets .

How can molecular dynamics simulations guide structure-based inhibitor design for A. baumannii chorismate synthase?

Molecular dynamics (MD) simulations offer powerful insights for structure-based inhibitor design targeting A. baumannii chorismate synthase:

• Binding pocket characterization: Extended MD simulations (50+ ns) reveal transient binding pocket conformations not observable in static crystal structures, identifying cryptic binding sites and allosteric regions .

• Water network mapping: MD simulations identify conserved water molecules in the active site that mediate enzyme-substrate interactions and can be displaced by inhibitors for enhanced binding affinity .

• Ligand optimization guidance: Analyzing protein-ligand interaction trajectories from MD simulations helps identify optimal functional groups and scaffold modifications for improved binding, as demonstrated with phytochemical derivatives .

• Residence time prediction: MD-based approaches like Markov State Modeling can estimate inhibitor residence times, which often correlate better with in vivo efficacy than equilibrium binding constants .

• Binding energy calculations: MM/PBSA approaches applied to MD trajectories provide more accurate binding energy estimates than static docking, helping prioritize compounds for experimental validation .

Implementation of this approach has successfully identified that gallic acid demonstrates superior binding profiles compared to caffeic acid and o-coumaric acid against bacterial chorismate synthase, with binding energies confirmed by isothermal calorimetry .

What are the challenges and solutions in developing aroC inhibitors that maintain efficacy against evolving Acinetobacter strains?

Developing aroC inhibitors effective against evolving Acinetobacter strains presents several challenges with corresponding solution strategies:

Challenges:

• Target site mutations: Natural selection may favor aroC variants with altered inhibitor binding sites while maintaining catalytic function .

• Efflux pump upregulation: A. baumannii readily upregulates efflux pumps that can expel antibiotics before they reach intracellular targets .

• Biofilm formation: Mature biofilms create physical barriers and altered metabolic states that reduce inhibitor effectiveness .

• Metabolic bypass: Alternative pathways may emerge under selective pressure to compensate for inhibited chorismate synthesis .

Solutions:

• Multi-target inhibitor design: Developing dual-action inhibitors that simultaneously target multiple enzymes in the shikimate pathway (e.g., both aroC and aroA) reduces the likelihood of resistance development .

• Efflux pump inhibitor co-administration: Combining aroC inhibitors with efflux pump inhibitors can maintain effective intracellular concentrations .

• Penetration-optimized molecules: Designing inhibitors with physicochemical properties that facilitate bacterial membrane penetration and minimize efflux substrate recognition .

• Structure-activity relationship mapping: Developing diverse inhibitor libraries that maintain activity against predicted resistance-conferring mutations based on comprehensive structural analysis .

• Combination therapy approaches: Using aroC inhibitors in combination with existing antibiotics with different mechanisms of action to create synergistic effects and reduce resistance development risk .

These approaches collectively address the adaptability of A. baumannii while exploiting the essential nature of the chorismate pathway .

What are common pitfalls when expressing recombinant A. baumannii chorismate synthase and how can they be addressed?

Common expression pitfalls and their solutions include:

• Inclusion body formation: Reduce induction temperature to 16°C and decrease IPTG concentration to 0.1-0.3 mM to promote proper folding .

• Low yield: Optimize codon usage for E. coli expression through synthetic gene design or use specialized strains containing rare tRNAs (e.g., Rosetta or CodonPlus) .

• Proteolytic degradation: Include protease inhibitors during lysis and purification steps and consider using E. coli strains with reduced protease activity (e.g., BL21) .

• Loss of activity during purification: Maintain reducing conditions throughout purification and add stabilizers such as glycerol (10-15%) and FMN cofactor (10 μM) to all buffers .

• Aggregation during storage: Filter purified protein through a 0.22 μm membrane before storage, add 1-5 mM DTT, and store at -80°C in small aliquots to prevent freeze-thaw cycles .

Addressing these challenges systematically can significantly improve the quality and quantity of recombinant A. baumannii chorismate synthase for downstream applications .

How can researchers troubleshoot inconclusive results in aroC inhibition assays?

When troubleshooting inconclusive inhibition assay results, researchers should:

• Verify enzyme quality: Confirm enzyme activity before inhibition testing using positive controls and ensure protein has not degraded through SDS-PAGE analysis .

• Evaluate compound properties: Test for compound solubility in assay buffers, potential aggregation, and intrinsic fluorescence or absorbance that might interfere with assay readouts .

• Rule out non-specific inhibition: Include detergent controls (0.01% Triton X-100) to identify promiscuous aggregation-based inhibitors that give false positives .

• Assess time-dependent effects: Investigate potential time-dependent inhibition by varying pre-incubation times of enzyme with inhibitor before substrate addition .

• Validate orthogonally: Confirm promising results using multiple assay formats (e.g., both spectrophotometric and HPLC-based methods) to rule out assay-specific artifacts .

• Check for redox cycling compounds: Some compounds may interfere with the redox state of the FMN cofactor; control experiments with varying FMN concentrations can identify such false positives .

These systematic troubleshooting approaches help distinguish true inhibitors from artifacts and provide more reliable data for structure-activity relationship development .

How might phytochemicals be optimized as aroC inhibitors based on binding studies?

Phytochemical optimization as aroC inhibitors can follow several strategic approaches based on binding studies:

• Core scaffold modification: Gallic acid, which shows superior binding affinity to chorismate synthase, provides a core structure that can be modified to enhance binding properties . Molecular dynamics simulations indicate that its three hydroxyl groups form critical hydrogen bonds with the enzyme active site .

• Pharmacophore-based design: Comparative analysis of caffeic acid, gallic acid, and o-coumaric acid binding profiles reveals essential interaction patterns that can guide rational design of hybrid molecules incorporating optimal features from each compound .

• Solubility-activity balance: While maintaining the critical phenolic hydroxyl groups that contribute to binding affinity, modifying other positions can improve pharmacokinetic properties without compromising inhibitory activity .

• Binding site targeting: Structure-guided modifications can specifically target unique features of the A. baumannii chorismate synthase binding pocket, enhancing selectivity over other bacterial homologs .

• Prodrug approaches: Converting these phytochemicals to prodrug forms that are activated within bacterial cells could improve cell penetration while maintaining target engagement .

This optimization process should be guided by iterative cycles of computational modeling, synthesis, and experimental validation to systematically improve both potency and specificity .

What is the potential for developing combination therapies targeting multiple enzymes in the shikimate pathway?

Developing combination therapies targeting multiple enzymes in the shikimate pathway offers significant promise for treating resistant Acinetobacter infections:

• Synergistic inhibition: Simultaneously targeting chorismate synthase (aroC) alongside other essential enzymes such as PSCVT (aroA) and shikimate kinase (aroK) creates synergistic effects that can overcome partial resistance to individual inhibitors .

• Resistance mitigation: Multi-target approaches significantly reduce the probability of resistance development, as bacteria would need to simultaneously develop mutations in multiple essential genes . This approach has proven successful in the folate pathway with trimethoprim-sulfamethoxazole combination therapy .

• Reduced dosing: Synergistic effects often allow for lower dosages of each individual inhibitor, potentially reducing toxicity concerns while maintaining efficacy .

• Complementary pharmacokinetics: Inhibitors with different physicochemical properties may achieve better tissue distribution collectively than single agents .

• Cross-species effectiveness: The conserved nature of the shikimate pathway across many Gram-negative pathogens suggests that such combination approaches could have broad-spectrum activity against multiple drug-resistant organisms beyond just Acinetobacter .

Experimental studies have already validated the in vivo essentiality of aroA, aroC, and aroK in A. baumannii infection models, providing strong support for this multi-target approach .

How can structural information from chorismate synthase be integrated with systems biology approaches to predict resistance mechanisms?

Integrating structural information with systems biology approaches enables more sophisticated prediction of resistance mechanisms:

• Resistance mutation prediction: Combining protein structural analysis with genomic data from clinical isolates allows identification of naturally occurring polymorphisms in the aroC gene that might predispose to resistance development .

• Metabolic network modeling: Flux balance analysis incorporating chorismate synthase inhibition can predict potential metabolic adaptations and bypass mechanisms that might emerge under selective pressure .

• Multi-omics integration: Correlating transcriptomic, proteomic, and metabolomic changes in response to chorismate synthase inhibition helps identify compensatory mechanisms and potential co-targets to prevent resistance .

• Evolutionary trajectory modeling: Using directed evolution experiments with structural analysis of evolved variants can identify likely resistance mutations before they emerge clinically .

• Machine learning prediction: Training algorithms on combined structural and systems data can identify non-obvious relationships between structural features, metabolic networks, and resistance potential .

This integrated approach provides a more comprehensive understanding of potential resistance mechanisms and informs proactive strategies to develop inhibitors less susceptible to resistance development .