Recombinant Synechocystis sp. Thiamine-phosphate synthase (thiE)

What is thiamine-phosphate synthase (thiE) and what is its role in Synechocystis sp.?

Thiamine-phosphate synthase (thiE) is a crucial enzyme in the thiamine biosynthesis pathway in Synechocystis sp. The enzyme catalyzes the substitution of pyrophosphate of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-PP) by 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate (Thz-P) to yield thiamin phosphate, which is subsequently phosphorylated to thiamin pyrophosphate (TPP), an essential cofactor in various metabolic processes . Thiamine biosynthesis is particularly important for cyanobacteria like Synechocystis sp., which are photosynthetic prokaryotes with unique physiological traits requiring thiamine-dependent enzymes .

How can I express recombinant thiE from Synechocystis sp. in a heterologous system?

Recombinant thiE from Synechocystis sp. can be successfully expressed in Escherichia coli using established molecular cloning techniques. The gene coding for thiE should be PCR-amplified from Synechocystis sp. genomic DNA with appropriate primers containing restriction sites, followed by ligation into a suitable expression vector (such as pET series vectors). For optimal expression, consider the following methodological approach:

• Clone the thiE gene into a vector with an inducible promoter (such as T7)

• Transform into an E. coli expression strain (BL21(DE3) or similar)

• Culture in LB or similar rich medium at 37°C until mid-log phase

• Induce protein expression with IPTG (typically 0.1-1.0 mM)

• Harvest cells after 3-6 hours of induction or after overnight expression at lower temperatures (16-25°C)

Similar to other recombinant proteins from Synechocystis, the expressed apoprotein is likely to be soluble and can be purified using affinity chromatography if tagged appropriately .

What are the structural characteristics of thiamine-phosphate synthase from Synechocystis sp.?

While the specific crystal structure of Synechocystis sp. thiamine-phosphate synthase has not been fully characterized in the provided research materials, comparative analysis with related enzymes suggests it likely belongs to the family of thiamine phosphate synthases with conserved structural features. Based on homology modeling approaches similar to those used for Mycobacterium tuberculosis TPS, Synechocystis thiE likely contains a substrate binding groove with specific residues that determine its catalytic efficiency .

The protein likely forms dimers in solution, similar to other bacterial thiamine-phosphate synthases and other recombinant proteins from Synechocystis, which tend to form dimers in vitro . The enzyme's active site would contain conserved residues responsible for binding the HMP-PP and Thz-P substrates, though specific amino acid compositions may differ from related enzymes in other organisms, potentially affecting substrate affinity and catalytic rates.

How does the substrate binding site of Synechocystis thiE compare to that of other bacterial thiamine-phosphate synthases?

The substrate binding site of thiamine-phosphate synthase shows interesting variations across bacterial species. While specific data for Synechocystis thiE is limited in the provided materials, comparative analysis with other bacterial TPSs provides valuable insights.

In Mycobacterium tuberculosis TPS, the substrate binding groove is notably narrower and shallower compared to TPS from Pyrococcus furiosus and Bacillus subtilis. This structural difference is attributed to three specific residues: Cys 139, Phe 174, and Arg 194 in M. tuberculosis, which replace shorter chain residues (Gly, Val, and Gly/Ser, respectively) in other bacterial species .

Synechocystis thiE likely exhibits species-specific variations in these key residues that would affect its substrate binding properties and catalytic efficiency. These structural differences may reflect adaptations to the unique metabolic requirements of cyanobacteria and their photosynthetic lifestyle .

What experimental approaches can be used to measure the kinetic parameters of recombinant Synechocystis thiE?

To determine the kinetic parameters of recombinant Synechocystis thiE, researchers should implement a multi-method approach:

• Enzymatic Assay Design:

  • Direct assay: Monitor the formation of thiamine phosphate using HPLC or coupled spectrophotometric assays

  • Indirect assay: Measure the release of pyrophosphate using enzymes that couple PPi hydrolysis to NAD+ reduction, followed by spectrophotometric detection at 340 nm

• Steady-State Kinetics Protocol:

  • Vary the concentration of HMP-PP (0.1-10× Km) while maintaining saturating Thz-P

  • Vary the concentration of Thz-P (0.1-10× Km) while maintaining saturating HMP-PP

  • Plot initial velocities against substrate concentrations to generate Michaelis-Menten curves

  • Determine Km, Vmax, and kcat using non-linear regression analysis

• Inhibition Studies:

  • Test product inhibition patterns with thiamine phosphate

  • Analyze dead-end inhibition with substrate analogs

  • Apply different inhibition models (competitive, uncompetitive, mixed) to determine inhibition constants

• Temperature and pH Dependence:

  • Measure activity across pH range (6.0-9.0) to determine optimum pH

  • Assess activity at various temperatures (20-50°C) to determine temperature optimum and calculate activation energy

This methodological approach follows experimental design principles that ensure validity and reliability while controlling for potential confounding variables .

How can I develop a high-throughput screening assay for inhibitors of Synechocystis thiE?

Developing a high-throughput screening (HTS) assay for Synechocystis thiE inhibitors requires careful experimental design that balances sensitivity, specificity, and throughput. A methodological approach based on successful screening efforts for M. tuberculosis TPS can be adapted:

• Primary Assay Development:

  • Implement a fluorescence-based or colorimetric detection method for thiamine phosphate production or pyrophosphate release

  • Optimize in 384-well microplate format with reaction volumes of 20-50 μL

  • Include suitable positive controls (known inhibitory compounds) and negative controls (DMSO vehicle)

• Assay Validation Protocol:

  • Determine Z' factor across multiple plates to ensure statistical reliability (aim for Z' > 0.5)

  • Assess DMSO tolerance (typically up to 1-2% v/v)

  • Evaluate day-to-day and plate-to-plate variability

• Compound Library Screening Strategy:

  • Screen at single concentration initially (10-50 μM)

  • Identify hits showing >50% inhibition

  • Confirm hits with dose-response curves (IC50 determination)

• Secondary Assays:

  • Counter-screen against related enzymes to determine selectivity

  • Evaluate cytotoxicity against mammalian cell lines

  • Assess antimicrobial activity against Synechocystis sp.

The virtual screening approach used for MtTPS, where the NCI diversity set II was screened against a homology model of the enzyme, identified several compounds with IC50 values ranging from 20-100 μg/ml . A similar computational approach could be employed for Synechocystis thiE to pre-select compounds with higher likelihood of inhibitory activity.

What purification strategy is most effective for obtaining high-purity recombinant Synechocystis thiE?

For high-purity recombinant Synechocystis thiE, a multi-step purification strategy is recommended:

• Affinity Chromatography (Primary Purification):

  • His-tagged thiE: Use Ni-NTA affinity chromatography with imidazole gradient elution (20-250 mM)

  • GST-tagged thiE: Use glutathione sepharose with reduced glutathione elution

  • Apply this step directly to cleared cell lysate in appropriate buffer (typically phosphate or Tris-based, pH 7.5-8.0, with 100-300 mM NaCl)

• Ion Exchange Chromatography (Secondary Purification):

  • Determine theoretical pI of Synechocystis thiE

  • For pI < 7: Use anion exchange (Q Sepharose) at pH > pI + 1

  • For pI > 7: Use cation exchange (SP Sepharose) at pH < pI - 1

  • Elute with linear salt gradient (0-1 M NaCl)

• Size Exclusion Chromatography (Polishing Step):

  • Use Superdex 75 or Superdex 200 columns depending on molecular weight

  • Buffer composition typically 50 mM Tris-HCl, pH 7.5, 150 mM NaCl

  • This step also determines oligomeric state of the protein

Based on experience with other recombinant proteins from Synechocystis, maintaining moderate salt concentrations (>100 mM) is important to prevent protein aggregation, as these proteins tend to form dimers in vitro and may aggregate under low salt conditions .

How can I assess the impact of mutations in the active site of Synechocystis thiE on enzyme activity?

To systematically assess the impact of active site mutations on Synechocystis thiE activity, implement this methodological approach:

• Rational Mutation Design:

  • Identify conserved residues through multiple sequence alignment of thiE from various species

  • Create a homology model of Synechocystis thiE to predict key catalytic and substrate-binding residues

  • Design mutations: conservative (similar properties) and non-conservative (different properties)

  • Consider mutations corresponding to those studied in other TPS enzymes, such as the key residues identified in M. tuberculosis TPS (equivalents to Cys 139, Phe 174, and Arg 194)

• Site-Directed Mutagenesis Protocol:

  • Use PCR-based site-directed mutagenesis with appropriate primers

  • Verify mutations by DNA sequencing

  • Express and purify mutant proteins using identical conditions as wild-type

• Kinetic Parameter Determination:

  • Measure Km and kcat for both substrates (HMP-PP and Thz-P)

  • Calculate catalytic efficiency (kcat/Km) for each substrate

  • Compare parameters to wild-type enzyme

• Structural Impact Assessment:

  • Perform circular dichroism to assess secondary structure changes

  • Use thermal shift assays to determine stability differences

  • If possible, obtain crystal structures of key mutants

This approach follows sound experimental design principles , controlling for variables by maintaining identical expression, purification, and assay conditions between wild-type and mutant proteins.

What are the recommended conditions for storing purified recombinant Synechocystis thiE to maintain stability and activity?

Maintaining the stability and activity of purified recombinant Synechocystis thiE requires careful attention to storage conditions. Based on protocols for similar enzymes and other recombinant Synechocystis proteins, the following recommendations are provided:

• Short-term Storage (1-2 weeks):

  • Temperature: 4°C

  • Buffer composition: 50 mM Tris-HCl or phosphate buffer, pH 7.5-8.0

  • Salt concentration: 150-300 mM NaCl (important to prevent aggregation)

  • Additives: 1-5 mM DTT or 0.5-2 mM TCEP to maintain reduced state of cysteine residues

  • Protein concentration: 1-5 mg/ml

• Long-term Storage (months to years):

  • Temperature: -80°C (preferred) or -20°C

  • Add cryoprotectant: 10-20% glycerol or 5-10% sucrose

  • Aliquot in small volumes to avoid repeated freeze-thaw cycles

  • Flash-freeze in liquid nitrogen before transferring to -80°C storage

• Stability Assessment Protocol:

  • Test enzyme activity after various storage times (0, 1, 2, 4, 8 weeks)

  • Compare different storage conditions side-by-side

  • Document activity retention as percentage of initial activity

• Recommended Storage Formulations:

As recombinant proteins from Synechocystis tend to form dimers in vitro and aggregate under low salt conditions , maintaining adequate salt concentration is particularly important for preserving the native state of the enzyme.

How can I investigate the role of thiE in Synechocystis sp. using genetic manipulation approaches?

Investigating the role of thiE in Synechocystis sp. through genetic manipulation requires a systematic approach leveraging the natural transformability of this cyanobacterium:

• Gene Knockout Strategy:

  • Design homologous recombination constructs with antibiotic resistance cassette flanked by thiE upstream and downstream regions (500-1000 bp each)

  • Transform Synechocystis sp. with the knockout construct

  • Select transformants on antibiotic-containing media

  • Verify complete segregation through PCR and Southern blotting

  • Supplement growth media with thiamine if the mutation is lethal

• Complementation Analysis:

  • Express wild-type thiE under control of an inducible promoter (such as PnrsB or Ptrc)

  • Introduce into knockout strain and assess phenotype restoration

  • Use plasmid vectors specific for Synechocystis such as those available through CyanoSource

• Conditional Knockdown Approach:

  • Implement CRISPR interference (CRISPRi) targeting thiE

  • Design sgRNA targeting the non-template strand of thiE

  • Express dCas9 and sgRNA under inducible promoters

  • Monitor thiE expression and resulting phenotypes at different repression levels

• Phenotypic Characterization:

  • Growth rate assessment in various conditions (normal light, high light, nutrient limitation)

  • Metabolomics analysis focusing on thiamine and related metabolites

  • Transcriptomics to identify compensatory responses

  • Stress response analysis (oxidative, temperature, etc.)

This methodological approach allows for comprehensive analysis of thiE function while accounting for potential essentiality of the gene for Synechocystis sp. survival. The CyanoSource resource mentioned in the literature provides valuable tools for genetic manipulation of Synechocystis sp. .

What techniques can be used to characterize the interaction between recombinant Synechocystis thiE and its substrates?

Characterizing interactions between recombinant Synechocystis thiE and its substrates requires multiple biophysical and biochemical approaches for a comprehensive understanding:

• Isothermal Titration Calorimetry (ITC):

  • Directly measures thermodynamic parameters of binding

  • Protocol: Titrate substrate (HMP-PP or Thz-P) into purified thiE solution

  • Parameters determined: Binding affinity (Kd), enthalpy (ΔH), entropy (ΔS), and stoichiometry

  • Buffer conditions: Typically 50 mM phosphate or HEPES, pH 7.5, 100-150 mM NaCl

• Surface Plasmon Resonance (SPR):

  • Provides real-time binding kinetics

  • Immobilize His-tagged thiE on NTA sensor chip

  • Flow substrate solutions at various concentrations

  • Determine association (kon) and dissociation (koff) rate constants

• Fluorescence-based Assays:

  • Intrinsic tryptophan fluorescence: Monitor changes upon substrate binding

  • Fluorescently labeled substrates: Measure binding through fluorescence polarization

  • FRET-based assays if suitable donor-acceptor pairs can be established

• X-ray Crystallography:

  • Co-crystallize thiE with substrates, substrate analogs, or products

  • Determine atomic resolution structures of enzyme-ligand complexes

  • Identify specific amino acid interactions with substrates

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Compare deuterium uptake patterns in free enzyme versus substrate-bound forms

  • Identify regions with altered solvent accessibility upon binding

  • Provide insights into conformational changes induced by substrate binding

By combining multiple techniques, researchers can develop a comprehensive model of substrate recognition and binding by Synechocystis thiE, which follows sound experimental design principles ensuring validity and reliability of the results .

How does the activity of recombinant Synechocystis thiE compare with the enzyme from other cyanobacterial species?

Comparative analysis of thiE activity across cyanobacterial species provides valuable insights into evolutionary adaptations of thiamine biosynthesis. While specific comparative data for cyanobacterial thiE enzymes is limited in the provided literature, a methodological approach for such comparison can be outlined based on established enzymatic characterization techniques:

• Standardized Expression and Purification:

  • Clone thiE genes from multiple cyanobacterial species (e.g., Synechocystis sp. PCC 6803, Synechococcus sp., Anabaena sp.)

  • Express under identical conditions using the same expression system

  • Purify using identical protocols to ensure comparable preparations

• Kinetic Parameter Comparison:

  • Determine Km and kcat for both substrates (HMP-PP and Thz-P) under identical assay conditions

  • Calculate catalytic efficiency (kcat/Km) for each substrate

  • Measure pH and temperature optima for each enzyme

• Comparative Analysis of Structural Features:

  • Generate homology models for each enzyme

  • Compare active site architecture and substrate binding regions

  • Analyze potential differences in key residues similar to those identified in other TPS comparisons

• Expected Patterns Based on Related Research:

  • Enzymes from different cyanobacterial species likely exhibit variations in catalytic efficiency reflecting their ecological niches

  • Differences may correlate with growth rates and metabolic requirements

  • The TM arrangement differences observed between Synechocystis (complicated, disparate patterns) and Synechococcus (orderly sheets parallel to PM) suggest potential physiological adaptations that might be reflected in enzyme kinetics

This comparative approach would provide valuable insights into how thiE function has evolved across cyanobacterial species with different physiological adaptations and ecological niches.

What are common challenges in expressing and purifying recombinant Synechocystis thiE and how can they be addressed?

Researchers often encounter several challenges when working with recombinant Synechocystis thiE. Based on experiences with other recombinant proteins from Synechocystis and related organisms, the following troubleshooting approaches are recommended:

• Expression Challenges:

• Purification Challenges:

• Activity Challenges:

• Methodological Refinement Approach:

Based on the observation that recombinant Synechocystis proteins tend to form dimers in vitro and aggregate under low salt conditions , special attention should be paid to buffer composition during purification and storage. Additionally, the thiE enzyme may exhibit specific requirements for activity that should be systematically investigated through buffer optimization experiments.

How can I optimize the in vitro reaction conditions for maximum Synechocystis thiE activity?

Optimizing in vitro reaction conditions for maximum Synechocystis thiE activity requires systematic parameter testing. The following methodological approach is recommended:

• Buffer System Optimization:

  • Test different buffers at constant pH (7.5):

    • Tris-HCl (50-100 mM)

    • HEPES (50-100 mM)

    • Phosphate buffer (50-100 mM)

    • MOPS (50-100 mM)

  • Determine optimal pH range (typically 6.5-9.0) using the best buffer

• Salt and Ionic Strength:

  • Test NaCl concentration range (0-500 mM)

  • Evaluate effects of different salts (KCl, NH₄Cl) at equivalent ionic strengths

  • Based on observations with other Synechocystis proteins, maintain >100 mM salt to prevent aggregation

• Metal Ion Requirements:

  • Screen divalent cations (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺) at 1-10 mM

  • Test EDTA to determine if metal ions are inhibitory

  • Combine optimal metal ion with chelator titration to determine precise requirements

• Reducing Agent Optimization:

  • Compare different reducing agents (DTT, β-mercaptoethanol, TCEP) at various concentrations

  • Determine if enzyme activity is sensitive to oxidation

• Temperature and Stability Parameters:

  • Determine temperature optimum (typically 25-45°C for cyanobacterial enzymes)

  • Assess thermal stability at optimal temperature

  • Evaluate freeze-thaw stability

The optimal reaction conditions determined through this systematic approach can be summarized in a table format:

This systematic optimization approach follows sound experimental design principles , controlling for variables by changing only one parameter at a time and then combining optimal conditions.

How can structural biology approaches contribute to understanding Synechocystis thiE catalytic mechanism?

Advanced structural biology techniques offer powerful tools for elucidating the catalytic mechanism of Synechocystis thiE. A comprehensive research strategy would include:

• X-ray Crystallography:

  • Solve structures of:

    • Apo-enzyme (substrate-free)

    • Enzyme-substrate complexes (with HMP-PP or Thz-P)

    • Enzyme-product complex (with thiamine phosphate)

    • Catalytic intermediates (if possible)

  • The solubility and stability properties of recombinant Synechocystis proteins make them amenable to crystallization attempts

• Cryo-Electron Microscopy:

  • Particularly valuable if the enzyme forms larger oligomeric structures

  • Can capture different conformational states during catalysis

  • Complementary to crystallographic studies

• NMR Spectroscopy:

  • Solution-state structural studies

  • Dynamics investigations to identify mobile regions

  • Chemical shift perturbation experiments to map substrate binding

• Time-Resolved Studies:

  • Mix-and-quench approaches to trap reaction intermediates

  • Time-resolved crystallography using synchrotron radiation

  • Temperature-jump methods coupled with spectroscopic detection

• Computational Approaches:

  • Molecular dynamics simulations to model substrate binding and catalysis

  • QM/MM methods to calculate reaction energy barriers

  • Docking studies to identify potential inhibitor binding modes

This multi-technique approach would provide insights into:

• Conformational changes upon substrate binding

• Specific residues involved in substrate recognition and catalysis

• Reaction coordinate and transition states

• Potential allosteric regulation mechanisms

The favorable properties of recombinant Synechocystis proteins noted in the literature, such as solubility and ease of purification , suggest that thiE would be a good candidate for these structural studies.

What are the implications of thiE research for understanding cyanobacterial metabolism and potential biotechnological applications?

Research on Synechocystis thiE extends beyond basic enzymology, offering broader insights into cyanobacterial metabolism and potential applications:

• Metabolic Network Integration:

  • Thiamine phosphate synthesis represents a critical node in cyanobacterial metabolism

  • Understanding thiE regulation provides insights into how cyanobacteria balance energy production (photosynthesis) with vitamin biosynthesis

  • Connections between thiamine metabolism and unique cyanobacterial features such as thylakoid membrane (TM) organization

• Stress Response Mechanisms:

  • Thiamine pyrophosphate (TPP) is essential for enzymes involved in carbon metabolism and oxidative stress response

  • thiE activity likely adjusts to environmental stressors (light intensity, nutrient availability)

  • Potential role in cyanobacterial adaptations to changing environments

• Synthetic Biology Applications:

  • Engineering cyanobacteria with modified thiE for enhanced thiamine production

  • Development of biosensors using thiE as a component

  • Utilization in cell-free systems for biocatalysis

• Drug Discovery Potential:

  • While not a pathogen, Synechocystis thiE research provides insights for targeting related enzymes in pathogenic organisms

  • The approach used for M. tuberculosis TPS inhibitor identification could be adapted for other bacterial pathogens

  • Cyanobacterial thiE could serve as a more experimentally tractable model system

• Ecological Implications:

  • Thiamine production by cyanobacteria impacts aquatic food webs

  • Understanding thiE regulation could help predict ecosystem responses to environmental changes

  • Potential connections to harmful algal bloom dynamics

The unique physiological characteristics of Synechocystis, including its complex thylakoid membrane arrangements compared to other cyanobacteria like Synechococcus , suggest that thiamine metabolism may have specialized adaptations in this organism that warrant further investigation.

How might CRISPR-based technologies be applied to study thiE function in Synechocystis sp.?

CRISPR-based technologies offer powerful approaches for investigating thiE function in Synechocystis sp., with methodological considerations tailored to this cyanobacterium:

These approaches can be implemented using the valuable resources for Synechocystis genetic manipulation mentioned in the literature, such as CyanoSource, which provides a mutant library and plasmid resources specifically for this organism . The methodological design follows sound experimental principles , ensuring appropriate controls and validation steps throughout the investigation.