Recombinant Geobacter sulfurreducens Chorismate synthase (aroC)

What is the role of chorismate synthase (aroC) in the shikimate pathway of Geobacter sulfurreducens?

Chorismate synthase (EC 4.2.3.5) catalyzes the final step in the seven-step shikimate pathway, which is critical for aromatic amino acid biosynthesis. This enzyme specifically converts 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate through a 1,4-trans elimination reaction . In G. sulfurreducens, aroC (gene locus GSU1242) produces chorismate as a crucial precursor for the synthesis of phenylalanine, tyrosine, and tryptophan .

The methodological approach to study this pathway involves:

• Metabolic flux analysis using isotope-labeled precursors

• Gene knockout experiments to confirm essentiality

• Comparative genomics to identify all components of the shikimate pathway in G. sulfurreducens

• Enzyme activity assays to characterize reaction kinetics in varying conditions

How does the structure of G. sulfurreducens aroC compare to chorismate synthase in other bacterial species?

Chorismate synthase adopts a homotetrameric structure with a unique beta-alpha-beta sandwich fold. Each monomer consists of 9 alpha helices and 18 beta strands, with one FMN molecule non-covalently bound to each monomer . While no G. sulfurreducens-specific structural data is presented in the search results, the high degree of sequence conservation among chorismate synthases (360-400 amino acids) suggests similar structural features .

Comparative analysis methodology would include:

• Homology modeling of G. sulfurreducens aroC based on solved structures (PDB entries 1Q1L, 1QXO, 1R52, etc.)

• Structural alignment focusing on FMN-binding regions

• Analysis of active site architecture, particularly the positions of catalytic histidine residues

• Comparative electrostatic potential mapping of the enzyme surface

What is the significance of the FMN cofactor in G. sulfurreducens aroC catalytic activity?

Unlike most redox enzymes, chorismate synthase requires reduced flavin mononucleotide (FMNH2 or FADH2) as a cofactor despite no net redox change occurring in the reaction . This unusual cofactor requirement represents a mechanistic puzzle in enzyme catalysis.

The methodological investigation of this phenomenon involves:

• Site-directed mutagenesis of FMN-binding residues

• Spectroscopic monitoring of FMN redox states during catalysis

• Kinetic analysis with varying FMN concentrations and redox states

• Computational modeling of electron transfer mechanisms

• Stopped-flow spectroscopy to capture transient intermediates

Two invariant histidine residues (His17 and His106) are critical to this mechanism, with His106 protonating the monoanionic reduced FMN while His17 protonates the leaving phosphate group .

How is chorismate synthase activity measured in G. sulfurreducens?

Multiple complementary approaches can be employed to measure aroC activity:

• High-Resolution Mass Spectrometry (HR-MS): A coupled enzyme assay using purified recombinant EPSP synthase and chorismate synthase, with product detection via LC/HRMS. This method avoids interference from reaction components that affect spectrophotometric methods .

• Spectrophotometric Assays: Monitoring consumption of reduced FMN or formation of chorismate based on their characteristic absorption profiles.

• Coupled Enzyme Reactions: Systems involving downstream enzymes that use chorismate as a substrate, such as anthranilate synthase.

• Real-Time, High-Throughput Assays: Particularly useful for screening multiple G. sulfurreducens strain constructs simultaneously .

What are the mechanistic details of the aroC catalytic cycle, and how do structural elements contribute to function?

Chorismate synthase employs a unique catalytic mechanism involving two sequential eliminations:

• First, phosphate elimination occurs, assisted by proton transfer from a conserved histidine residue (His17) .

• Simultaneously, an electron is transferred from the reduced FMN to the substrate, generating radicals.

• The FMN radical rearranges, followed by hydrogen atom transfer from the substrate, eliminating both radicals.

• Finally, FMN re-tautomerizes to its active form by donating a proton to a second conserved histidine (His106) .

Methodological approaches to investigate this mechanism include:

• Quantum mechanical/molecular mechanical (QM/MM) calculations

• Transient kinetic measurements using stopped-flow spectroscopy

• Electron paramagnetic resonance (EPR) to detect radical intermediates

• Hydrogen-deuterium exchange mass spectrometry to probe protein dynamics

• X-ray crystallography of enzyme-substrate complexes at various reaction stages

How can molecular dynamics simulations and virtual screening accelerate the discovery of aroC inhibitors?

Computational approaches offer powerful tools for inhibitor discovery targeting chorismate synthase:

• Structure-Based Virtual Screening: Using the three-dimensional structure of aroC to screen large compound libraries .

• Molecular Dynamics Simulations: Analyzing protein-ligand complex stability over time (50+ ns simulations) .

• Binding Energy Calculations: Employing MM/PBSA approaches to estimate binding affinities .

• Experimental Validation: Using isothermal titration calorimetry (ITC) to confirm binding predictions .

This integrated approach has successfully identified promising phytochemical inhibitors (including gallic acid, caffeic acid, and o-coumaric acid) targeting chorismate synthase from other bacterial species . Similar methodologies could be applied to G. sulfurreducens aroC.

How is G. sulfurreducens aroC expression integrated with the organism's unique electron transfer mechanisms?

G. sulfurreducens possesses extraordinary electron transfer capabilities essential for its environmental adaptations. Understanding the integration between aromatic amino acid biosynthesis and electron transfer requires:

• Transcriptomic Analysis: Examining aroC expression under varying electron acceptor conditions (Fe(III) oxide, electrodes, etc.) .

• Metabolic Flux Analysis: Tracing carbon flow between central metabolism, aromatic amino acid synthesis, and electron transfer pathways.

• FMN Homeostasis Investigation: Examining how FMN reduction for aroC activity connects to broader cellular electron transfer networks.

• Regulatory Network Mapping: Identifying potential co-regulation between aroC and electron transfer genes.

G. sulfurreducens possesses sophisticated regulatory systems including cyclic nucleotide signaling (c-di-GMP, cGAMP) that influence electron transfer , which may indirectly affect aromatic amino acid metabolism.

What protocols are most effective for heterologous expression and purification of G. sulfurreducens aroC?

Based on successful approaches with related enzymes and G. sulfurreducens proteins, the following methodology is recommended:

• Vector Selection: RK2-based plasmids show superior stability in G. sulfurreducens compared to pBBR1 plasmids, remaining stable for 15+ generations without antibiotic selection .

• Promoter Engineering:

  • For constitutive expression: Native G. sulfurreducens promoters

  • For controlled expression: VanR-dependent system inducible by vanillate

• Expression Systems:

  • Homologous expression in G. sulfurreducens (preferred for functional studies)

  • Heterologous expression in E. coli BL21(DE3) for high protein yields

• Purification Strategy:

  • Initial capture via affinity chromatography (His-tag on Ni-NTA columns)

  • Secondary purification by anion exchange chromatography (Q-Sepharose)

  • Final polishing using size exclusion chromatography

• Activity Verification:

  • Spectrophotometric assays

  • Mass spectrometry-based activity assessment

How can researchers develop effective enzyme assays for G. sulfurreducens aroC?

Developing robust, reproducible assays requires consideration of several factors:

• Cofactor Handling: Since aroC requires reduced FMN, maintaining appropriate reducing conditions is essential. Methods include:

  • Chemical reduction with sodium dithionite

  • Enzymatic reduction using flavin reductase

  • Photoreduction techniques

• Assay Formats:

  • Direct Assays: Monitoring chorismate formation by HPLC or LC-MS/MS

  • Coupled Assays: Using EPSP synthase to generate substrate in situ

  • Spectrophotometric Methods: Tracking changes in absorbance associated with FMN oxidation state

• Validation Approaches:

  • Confirm linearity with respect to time and enzyme concentration

  • Establish substrate and cofactor saturation curves

  • Determine kinetic parameters (Km, kcat, kcat/Km)

  • Benchmark against known chorismate synthase enzymes

• High-Throughput Adaptations:

  • Miniaturization to microplate format

  • Development of fluorescence-based readouts

  • Integration with automated liquid handling systems

What conserved domains in G. sulfurreducens aroC contribute to its enzymatic function?

Chorismate synthase contains several functionally important domains that contribute to its catalytic mechanism:

Table 1: Key Functional Domains in Chorismate Synthase

Methodologies to investigate domain functions include:

• Domain swapping experiments between different chorismate synthases

• Alanine scanning mutagenesis of conserved residues

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Molecular dynamics simulations to analyze domain motions during catalysis

How does the substrate specificity of G. sulfurreducens aroC compare to that of other organisms?

Understanding substrate specificity differences requires:

• Kinetic Analysis: Determining Km and kcat values for EPSP and potential substrate analogs across multiple species.

• Structural Comparison: Analyzing substrate binding pocket architecture among chorismate synthases from different organisms.

• Substrate Analog Testing: Evaluating the enzyme's ability to process modified substrates.

• Protein Engineering: Creating chimeric enzymes by swapping substrate-binding regions between species.

• Evolutionary Analysis: Correlating sequence variations in substrate-binding regions with ecological niches or metabolic capabilities.

How has the aroC gene evolved within Geobacteraceae family members?

Investigating evolutionary patterns requires:

• Sequence Alignment: Comparing aroC sequences across Geobacteraceae and other bacterial families.

• Phylogenetic Analysis: Constructing trees to visualize evolutionary relationships.

• Selection Pressure Analysis: Calculating dN/dS ratios to identify conserved vs. variable regions.

• Genomic Context Examination: Analyzing gene neighborhood conservation across species.

• Horizontal Gene Transfer Assessment: Identifying potential gene acquisition events.

What is the druggability assessment of G. sulfurreducens aroC as an antimicrobial target?

The shikimate pathway represents an attractive antimicrobial target since it is absent in mammals . Comprehensive druggability assessment includes:

• Evolutionary Conservation Analysis: Determining conserved regions across bacterial species .

• Binding Pocket Identification: Using computational tools like SiteMap to evaluate binding sites .

• Physicochemical Property Evaluation: Assessing properties that favor small molecule binding .

• Comparison with Known Inhibitor Properties: Analyzing whether binding sites can accommodate drug-like molecules .

This systematic approach provides structural and molecular insights into whether aroC is a suitable target for developing broad-spectrum antibacterial drugs .

What are the main challenges in studying G. sulfurreducens aroC and how can they be addressed?

Table 2: Research Challenges and Potential Solutions

How can phytochemical inhibitors be developed as antimicrobial agents targeting G. sulfurreducens aroC?

Building on research with other bacterial chorismate synthases , a comprehensive approach includes:

• Virtual Screening Against Natural Product Libraries: Identifying candidates with favorable binding profiles to aroC models.

• Molecular Dynamics Simulation: Evaluating ligand stability in the binding pocket over extended timeframes.

• Structure-Activity Relationship Studies: Synthesizing derivatives of promising compounds (like caffeic acid, gallic acid) to improve potency and selectivity.

• Binding Validation: Using biophysical techniques like isothermal titration calorimetry to confirm interactions.

• Whole-Cell Testing: Evaluating antimicrobial activity against G. sulfurreducens and potential off-target effects.

• Delivery Optimization: Developing approaches to ensure inhibitor access to intracellular targets.

This integrated approach holds promise for developing novel antimicrobial compounds with specific activity against shikimate pathway enzymes.

How does aroC function contribute to G. sulfurreducens' unique environmental adaptations?

G. sulfurreducens possesses remarkable capabilities including metal reduction, electricity generation, and diverse carbon metabolism . Understanding aroC's role requires:

• Phenotypic Analysis: Comparing aroC mutants with wild-type strains under various environmental conditions.

• Transcriptomic Profiling: Examining aroC expression during biofilm formation, metal reduction, and electrode growth.

• Metabolomic Integration: Tracking aromatic compounds during environmental transitions.

• Synthetic Biology Approaches: Engineering aroC expression levels to assess impacts on G. sulfurreducens unique capabilities.

The vanillate-inducible expression system developed for G. sulfurreducens provides an excellent tool for such studies, allowing precise control of gene expression levels .

How can genetic tools be optimized for studying aroC function in G. sulfurreducens?

Recent advances in G. sulfurreducens genetic manipulation offer powerful approaches for aroC research:

• Scarless Genome Editing: Enables precise gene deletion or modification without polar effects .

• Stable Expression Vectors: RK2-based plasmids maintain stability without selection pressure .

• Inducible Systems: VanR-dependent promoters allow controlled gene expression .

• High-Throughput Screening: Real-time assays facilitate rapid assessment of multiple constructs .

These tools can be applied to create aroC variants, study regulatory elements, and examine pathway interconnections without the limitations of previous genetic systems.