Recombinant Thermus thermophilus 3-phosphoshikimate 1-carboxyvinyltransferase (aroA)

What is the role of 3-phosphoshikimate 1-carboxyvinyltransferase (PSCVT) in the shikimate pathway?

PSCVT, encoded by the aroA gene, catalyzes the transfer of the enolpyruvyl moiety from phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), forming 5-enolpyruvylshikimate-3-phosphate (EPSP) and inorganic phosphate. This represents a critical step in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in bacteria, fungi, and plants. The enzyme undergoes a significant conformational change from an open to closed form during substrate binding, which is critical for its catalytic function .

How should I design an expression system for recombinant Thermus thermophilus aroA?

For expression of thermostable T. thermophilus aroA, consider using a pET vector system in E. coli BL21(DE3) with the following modifications:

• Optimize codon usage for E. coli while maintaining the amino acid sequence

• Include a C-terminal His6-tag for purification, avoiding N-terminal tags that might interfere with folding

• Culture at 37°C until OD600 reaches 0.6-0.8, then induce with 0.5-1.0 mM IPTG

• Continue expression at 30°C for 4-6 hours to balance yield with proper folding

• For higher purity preparations, consider heat treatment at 65°C for 20 minutes post-lysis to precipitate E. coli proteins while retaining the thermostable T. thermophilus aroA

Validate expression using SDS-PAGE and Western blotting with anti-His antibodies, and confirm enzyme activity using a phosphate release assay with S3P and PEP substrates.

What purification protocol yields the highest activity for recombinant T. thermophilus aroA?

A multi-step purification protocol optimized for thermostable enzymes is recommended:

• Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and 20 mM imidazole

• Centrifuge at 20,000 × g for 30 minutes at 4°C

• Heat treatment: 65°C for 20 minutes to precipitate host proteins

• Ni-NTA affinity chromatography: Apply supernatant to pre-equilibrated column

• Wash with increasing imidazole concentrations (50 mM, 100 mM)

• Elute with 250 mM imidazole

• Size exclusion chromatography using Superdex 200 in 25 mM Tris-HCl pH 7.5, 150 mM NaCl

• Concentrate to 2-5 mg/ml and store with 10% glycerol at -80°C

This protocol typically yields >95% pure protein with specific activity of approximately 15-20 μmol/min/mg when assayed at 60°C.

How does the thermal stability of T. thermophilus PSCVT compare to mesophilic homologs, and what methods are optimal for measuring this difference?

T. thermophilus PSCVT demonstrates significantly higher thermal stability compared to mesophilic homologs. Differential Scanning Fluorimetry (DSF) reveals that unliganded T. thermophilus PSCVT exhibits a melting temperature (Tm) of approximately 53°C, which increases substantially to 61.7°C upon binding its substrate, shikimate 3-phosphate (S3P) . This 8°C shift indicates enhanced thermostability in the substrate-bound state.

For comparative thermal stability analysis, the following protocol is recommended:

When comparing thermal stability between T. thermophilus PSCVT and mesophilic homologs, it's essential to maintain identical buffer conditions (pH, ionic strength, additives) across all samples to ensure valid comparisons.

What structural modifications contribute to the thermostability of T. thermophilus aroA, and how can I investigate these experimentally?

Several structural features likely contribute to the thermostability of T. thermophilus aroA:

• Increased number of salt bridges

• Higher proportion of charged amino acids on the protein surface

• Reduced number of thermolabile residues

• Compact packing of hydrophobic core

• Increased proline content in loop regions

To investigate these features experimentally:

• Homology modeling and molecular dynamics simulations: Generate models at different temperatures to identify stabilizing interactions

• Site-directed mutagenesis: Target residues suspected to contribute to thermostability based on:

  • Surface charge clusters

  • Core packing residues

  • Loop regions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Measure local stability and flexibility differences between wild-type and mutants

• Differential scanning calorimetry (DSC): Quantify thermodynamic parameters (ΔH, ΔS, ΔG) of unfolding

• X-ray crystallography: Determine high-resolution structures in both open and closed conformations to visualize conformational changes during substrate binding, similar to the approach used for other T. thermophilus proteins

How can I design in vivo studies to evaluate the essentiality of aroA in T. thermophilus?

In vivo studies for aroA essentiality in T. thermophilus can be designed based on approaches used for similar enzymes in other organisms. The methodology should include:

• Gene disruption strategy:

  • Construct a plasmid containing a selectable marker (e.g., kanamycin resistance) flanked by DNA sequences homologous to regions upstream and downstream of the aroA gene

  • Transform into T. thermophilus using natural competence or electroporation

  • Select for transformants on media containing the appropriate antibiotic

• Validation of disruption:

  • PCR verification of gene disruption

  • RT-PCR to confirm absence of aroA transcript

  • Western blotting to verify absence of protein (if antibodies are available)

• Phenotypic analysis:

  • Growth curve analysis in rich vs. minimal media

  • Supplementation studies with aromatic amino acids

  • In vivo survival assays similar to those conducted for other essential genes

• Complementation studies:

  • Reintroduce aroA on a plasmid under control of an inducible promoter

  • Verify restoration of growth phenotype

  • Consider using the approach described for in vivo studies in the rat soft tissue infection model, adapting the protocol for T. thermophilus

What approaches can be used to study the conformational changes of T. thermophilus PSCVT during substrate binding?

The conformational transition from an open to closed form during substrate binding is a critical aspect of PSCVT function . To investigate this:

• X-ray crystallography:

  • Crystallize PSCVT in both apo and substrate-bound forms

  • Analyze structural differences, focusing on active site architecture

  • Consider soaking experiments with various ligands to capture intermediate states

• Small-angle X-ray scattering (SAXS):

  • Measure solution-state conformational changes

  • Analyze radius of gyration (Rg) changes upon substrate binding

  • Generate low-resolution envelope models for different states

• Förster resonance energy transfer (FRET):

  • Introduce fluorescent probes at strategic positions (via cysteine mutagenesis)

  • Monitor distance changes during substrate binding in real-time

  • Determine kinetics of conformational change

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Compare deuterium uptake profiles of apo and substrate-bound states

  • Identify regions with altered solvent accessibility

  • Map conformational dynamics onto structural models

• Molecular dynamics simulations:

  • Simulate transition between open and closed conformations

  • Calculate energy barriers for conformational changes

  • Identify key residues involved in the transition

How should enzyme kinetics experiments be designed for T. thermophilus aroA to account for its thermophilic nature?

When designing enzyme kinetics experiments for thermophilic T. thermophilus aroA:

• Temperature optimization:

  • Conduct initial velocity studies at multiple temperatures (50-80°C)

  • Determine temperature optima for catalytic efficiency (kcat/Km)

  • Ensure temperature stability of substrates throughout the assay duration

• Buffer considerations:

  • Use buffers with minimal temperature-dependent pH changes (e.g., HEPES)

  • Adjust pH at the assay temperature, not room temperature

  • Include thermostable additives for enzyme stabilization if needed

• Assay methods:

  • Continuous assays: Coupled enzyme systems must use thermostable coupling enzymes

  • Discontinuous assays: Quench reactions rapidly to prevent post-reaction activity

  • Consider using sealed capillary tubes or high-pressure vessels for temperatures above 80°C

• Data analysis:

  • Apply appropriate temperature corrections to extinction coefficients

  • Use Arrhenius plots to determine activation energy

  • Compare kinetic parameters across the temperature range to identify potential shifts in rate-limiting steps

What are the best approaches for studying inhibitor binding to T. thermophilus PSCVT?

To study inhibitor interactions with T. thermophilus PSCVT:

• Thermal shift assays:

  • Differential scanning fluorimetry (DSF) to measure Tm shifts upon inhibitor binding

  • Compare Tm shifts between substrate (S3P) and potential inhibitors

  • Use concentration gradients to determine binding affinity

• Enzyme inhibition assays:

  • Determine IC50 values at optimal enzyme temperature

  • Analyze inhibition patterns (competitive, non-competitive, uncompetitive)

  • Generate Dixon plots and Lineweaver-Burk plots for mechanism determination

• Isothermal titration calorimetry (ITC):

  • Measure binding thermodynamics (ΔH, ΔS, ΔG)

  • Determine binding stoichiometry

  • Conduct experiments at multiple temperatures to calculate ΔCp

• Surface plasmon resonance (SPR):

  • Real-time binding kinetics (kon, koff)

  • Test temperature dependence of binding interactions

  • Evaluate residence time of inhibitors at different temperatures

How can I design experiments to determine if T. thermophilus aroA is essential in vivo?

To determine if aroA is essential in T. thermophilus:

• Gene deletion approach:

  • Attempt construction of a clean deletion mutant using homologous recombination

  • If direct deletion fails, create a conditional mutant where expression is under control of an inducible promoter

  • Use a complementation system similar to the approach described for aroA mutants in A. baumannii

• Growth medium supplementation:

  • Test growth in minimal medium with and without aromatic amino acids

  • Determine if supplementation can rescue deletion phenotype

  • Quantify growth rates under various supplementation conditions

• In vivo survival assay:

  • Adapt the rat soft tissue infection model used for other essential genes

  • Compare bacterial counts over time between wild-type and aroA mutant strains

  • Analyze at appropriate temperatures for T. thermophilus growth

• Metabolomic analysis:

  • Profile metabolite changes in conditional mutants upon depletion

  • Focus on shikimate pathway intermediates

  • Identify potential metabolic bypass routes if present

How should I address inconsistencies in thermal stability data for T. thermophilus PSCVT?

When encountering discrepancies in thermal stability measurements:

• Standardize experimental conditions:

  • Ensure consistent protein concentration across methods

  • Verify buffer composition is identical between experiments

  • Maintain consistent scan rates for thermal ramping

• Method-specific considerations:

  • DSF: Test multiple dyes (SYPRO Orange, ThT) and concentrations

  • CD: Ensure sufficient signal-to-noise at higher temperatures

  • Activity assays: Account for substrate stability at elevated temperatures

• Data normalization approaches:

  • Use multiple reference points for baseline corrections

  • Apply protein-specific normalization factors based on molecular weight and extinction coefficients

  • Consider derivative analysis instead of raw data for transition midpoint determination

• Statistical validation:

  • Perform at least triplicate measurements

  • Apply appropriate statistical tests (ANOVA, t-tests) to evaluate significance of differences

  • Report confidence intervals alongside Tm values

What controls are essential when performing in vivo studies with T. thermophilus aroA mutants?

Essential controls for in vivo studies include:

• Genetic controls:

  • Wild-type parent strain (positive control)

  • Known viable deletion mutant in non-essential gene (procedure control)

  • Complemented aroA mutant (specificity control)

  • Similar approach to that used in the rat soft tissue infection model

• Growth condition controls:

  • Rich versus minimal media comparisons

  • Temperature range testing (standard and stress conditions)

  • Various carbon source utilization

• Phenotypic validation:

  • Verification of growth defects by multiple methods (plate count, optical density, ATP measurement)

  • Metabolite profiling to confirm pathway disruption

  • Protein expression verification by Western blot

• Experimental design considerations:

  • Include biological replicates (minimum n=3)

  • Conduct time-course analyses rather than endpoint measurements

  • Include appropriate statistical analyses similar to those used in comparable studies

How can I optimize crystallization conditions for T. thermophilus PSCVT structural studies?

For successful crystallization of T. thermophilus PSCVT:

• Initial screening strategy:

  • Sparse matrix screens at multiple temperatures (4°C, 18°C, 30°C)

  • Test both apo and substrate-bound forms

  • Vary protein concentration (5-15 mg/ml)

• Optimization approaches:

  • Fine-tune promising conditions by varying pH (±0.5 units), precipitant concentration (±5%), and additive concentrations

  • Consider seeding techniques from initial microcrystals

  • Test crystallization with and without His-tag (or after tag removal)

• Co-crystallization strategy:

  • For substrate complex: Incubate with S3P at 2-5× Km concentration

  • For inhibitor complexes: Use concentration at 3-5× IC50

  • Consider stability of complexes at crystallization temperature

• Data collection considerations:

  • Test multiple cryoprotectants (glycerol, ethylene glycol, sucrose at 15-25%)

  • Consider room-temperature data collection for thermostable proteins

  • Collect complete datasets with redundancy >4 to ensure data quality

How can comparative studies between T. thermophilus PSCVT and homologs from other species inform protein engineering efforts?

Comparative analysis between thermophilic and mesophilic PSCVT homologs provides valuable insights for protein engineering:

What methodologies can be used to study the RNA-binding properties of T. thermophilus proteins that might interact with aroA?

Although aroA encodes an enzyme rather than an RNA-binding protein, understanding potential RNA interactions in the broader T. thermophilus system can provide valuable research context. Based on methodologies used for other T. thermophilus proteins like Hera :

• Enhanced Cross-Linking and Immunoprecipitation (eCLIP):

  • Cross-link protein-RNA complexes in vivo using UV irradiation

  • Immunoprecipitate protein of interest with specific antibodies

  • Sequence bound RNAs to identify interaction partners

• RNA electrophoretic mobility shift assay (EMSA):

  • Test binding of purified protein to candidate RNA transcripts

  • Analyze complex formation through gel mobility shifts

  • Determine binding affinity through titration experiments

• RNA footprinting:

  • Use chemical or enzymatic probes to identify protected RNA regions

  • Map binding sites at nucleotide resolution

  • Compare footprints under various conditions (temperature, salt)

• Fluorescence anisotropy:

  • Label RNA with fluorescent dye

  • Measure changes in anisotropy upon protein binding

  • Determine binding constants at different temperatures

How might translational health research methodologies be applied to studies involving T. thermophilus aroA?

Translational research methodologies can bridge basic aroA enzyme studies with practical applications:

• Mixed methods approach:

  • Combine quantitative enzyme characterization with qualitative assessment of application potential

  • Integrate biochemical data with systems biology approaches

  • Apply similar mixed method designs as described for other translational health research

• Participatory research models:

  • Engage stakeholders (biotechnology industry, agricultural researchers) in study design

  • Use theoretical frameworks like the social-ecological model to contextualize enzyme applications

  • Develop research questions that address real-world implementation challenges

• Qualitative methodologies for application assessment:

  • Conduct focus groups with potential end-users of aroA-based technologies

  • Perform document analysis of current technological approaches

  • Use thematic analysis to identify barriers to implementation

• Translational indicators:

  • Define metrics for successful translation from basic research to application

  • Develop validation protocols for industrial or agricultural applications

  • Create frameworks for technology assessment similar to those used in health research

What statistical approaches are most appropriate for analyzing thermal stability data of T. thermophilus PSCVT?

For robust analysis of thermal stability data:

• Non-linear regression models:

  • Fit thermal denaturation curves to Boltzmann sigmoid equation

  • Use four-parameter logistic models for complex transitions

  • Apply Bayesian analysis for datasets with high variability

• Comparative statistical tests:

  • ANOVA with post-hoc tests for multiple condition comparisons

  • Paired t-tests for before/after substrate addition experiments

  • Non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) for non-normally distributed data

• Error analysis:

  • Report standard error of Tm determination

  • Use bootstrap resampling for confidence interval estimation

  • Perform sensitivity analysis for fitting parameters

• Data visualization:

  • Create thermal shift plots showing raw data points and fitted curves

  • Generate comparative bar charts with error bars for Tm values

  • Use statistical software (R, GraphPad Prism) for consistent analysis