Recombinant Methanothermobacter marburgensis Putative thymidylate synthase (thyA)

What is Methanothermobacter marburgensis Putative thymidylate synthase (thyA) and what is its biological significance?

Methanothermobacter marburgensis Putative thymidylate synthase (thyA) is an enzyme classified under EC 2.1.1.- from the archaeon Methanothermobacter marburgensis (also known as Methanobacterium thermoautotrophicum). Thymidylate synthase (TS) plays a critical role in DNA replication by catalyzing the synthesis of thymidylate (dTMP), which is essential for DNA biosynthesis .

The protein is of particular research interest because thymidylate synthases are highly conserved enzymes involved in cellular replication, making them important targets for functional studies and drug discovery. The enzyme from M. marburgensis, with UniProt accession number P80305, has been characterized as a putative thymidylate synthase .

How should Recombinant Methanothermobacter marburgensis Putative thymidylate synthase be properly stored and handled?

For optimal stability and activity retention, the recombinant protein should be stored at -20°C for regular use, or at -20°C to -80°C for extended storage. To minimize protein degradation, repeated freezing and thawing should be avoided. If working with the protein regularly, it is recommended to prepare working aliquots and store them at 4°C for up to one week .

For reconstitution, briefly centrifuge the vial prior to opening to bring the contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) before aliquoting and storing at -20°C/-80°C .

What are the best methods for purifying active Methanothermobacter marburgensis Putative thymidylate synthase from recombinant expression systems?

Purification of active thymidylate synthase requires careful consideration of buffer conditions and protein stability. Based on related archaeal protein purification protocols, the following method is recommended:

• Harvest recombinant cells expressing the protein.

• Resuspend cells in potassium phosphate buffer (pH 7.0).

• Lyse cells using a French pressure cell at 10^8 Pa or alternative methods such as sonication.

• Remove cell debris and membrane fraction by ultracentrifugation.

• Apply the supernatant to an appropriate affinity column based on the tag used during recombinant expression.

• Elute the protein with appropriate buffers.

• Further purify using size exclusion chromatography if needed.

• Determine protein concentration using the Bradford method with bovine serum albumin as the standard .

For quality control, analyze the purified protein using SDS-PAGE to confirm >85% purity, as is standard for recombinant preparations of this enzyme .

How can researchers design assays to measure the enzymatic activity of Recombinant Methanothermobacter marburgensis Putative thymidylate synthase?

Designing effective enzymatic assays for thymidylate synthase requires consideration of its catalytic function. Based on established methods for thymidylate synthase activity measurements, the following approaches are recommended:

• Spectrophotometric Assay: Monitor the conversion of 5,10-methylenetetrahydrofolate to dihydrofolate by measuring the increase in absorbance at 340 nm.

• Radiochemical Assay: Use radiolabeled substrates (either [5-³H]dUMP or [6-³H]dUMP) and measure the release of tritium into water during the reaction.

• Fluorescence-Based Assay: Utilize dansyl derivatives which have been shown to reveal bacterial specificity of thymidylate synthases .

When designing these assays, it's important to consider appropriate controls, including:

• Negative controls without enzyme

• Positive controls with commercially available thymidylate synthase

• Controls with known inhibitors of thymidylate synthase

Additionally, ensure that assay conditions (pH, temperature, buffer composition) are optimized for the archaeal enzyme, which may have different requirements than bacterial or eukaryotic thymidylate synthases.

What are the considerations for implementing site-directed mutagenesis studies on Methanothermobacter marburgensis Putative thymidylate synthase?

Site-directed mutagenesis is a powerful approach for understanding structure-function relationships in thymidylate synthase. Key considerations include:

A systematic approach might involve creating alanine substitutions at key residues, followed by more specific substitutions based on initial results.

How can researchers utilize Recombinant Methanothermobacter marburgensis Putative thymidylate synthase in comparative studies with other archaeal enzymes?

Comparative studies between archaeal enzymes can provide valuable insights into evolutionary relationships and specialized adaptations. For rigorous comparative studies:

• Selection of Comparison Targets: Include thymidylate synthases from diverse archaeal species, particularly those with different growth conditions or metabolic strategies. For example, compare with Methanosphaera stadtmanae thyA (Msp1237) .

• Parameter Standardization: Ensure consistent experimental conditions for all enzymes being compared, including buffer composition, pH, temperature, and substrate concentrations.

• Data Collection and Analysis: Collect comprehensive biochemical parameters (K​m, k​cat, temperature optima, pH profiles, inhibitor sensitivity) for each enzyme.

• Structural Comparisons: If structures are available, perform detailed structural alignments to identify conserved and divergent regions.

• Evolutionary Context: Interpret results in the context of the organisms' evolutionary history and environmental adaptations.

A suggested experimental design might include the following enzymes for comparison:

This approach would help identify adaptations specific to different environmental niches and metabolic strategies.

What are the challenges in crystallizing Recombinant Methanothermobacter marburgensis Putative thymidylate synthase and how can they be overcome?

Crystallizing archaeal proteins presents specific challenges due to their unique structural properties. For thymidylate synthase from M. marburgensis:

• Protein Purity and Homogeneity: Ensure >95% purity for crystallization attempts, which may require additional purification steps beyond the standard >85% purity .

• Buffer Optimization: Screen various buffer conditions, paying particular attention to salt concentration and pH, as archaeal proteins often have specific requirements reflecting their native environments.

• Ligand Co-crystallization: Consider co-crystallization with substrates, products, or inhibitors to stabilize the protein structure.

• Temperature Considerations: Since M. marburgensis is thermophilic, crystallization at elevated temperatures might yield better results.

• Surface Engineering: If initial crystallization attempts fail, consider surface entropy reduction or the creation of fusion constructs to promote crystal contacts.

• Alternative Approaches: If crystallization proves challenging, consider small-angle X-ray scattering (SAXS) or cryo-electron microscopy as alternative structural determination methods.

A systematic approach using sparse matrix screens followed by optimization of promising conditions is recommended.

How can researchers investigate the potential antimicrobial applications of targeting thymidylate synthase in methanogenic archaea?

Thymidylate synthase is an essential enzyme in DNA biosynthesis and has been successfully targeted for antimicrobial and anticancer drug development. For investigating antimicrobial potential against methanogenic archaea:

• Inhibitor Screening: Utilize both high-throughput screening and rational design approaches to identify compounds that selectively inhibit archaeal thymidylate synthase.

• Selectivity Analysis: Compare inhibition profiles against bacterial, archaeal, and human thymidylate synthases to identify compounds with selectivity for archaeal enzymes.

• Structure-Activity Relationships: Develop structure-activity relationships for promising inhibitors, focusing on compounds that exploit unique features of the archaeal enzyme.

• Dansyl Derivatives: Explore dansyl derivatives which have been shown to reveal bacterial specificity of thymidylate synthases to potentially develop archaeal-specific inhibitors.

• Whole-Cell Testing: Evaluate promising compounds for their ability to inhibit growth of methanogenic archaea in culture.

• Resistance Development: Assess the potential for resistance development through prolonged exposure experiments.

This research could have applications in controlling methanogenesis in various environments, including bioremediation and potential medical applications for archaeal intestinal commensals.

What are common issues in recombinant expression of Methanothermobacter marburgensis Putative thymidylate synthase and how can they be resolved?

Recombinant expression of archaeal proteins in heterologous systems often presents challenges:

• Poor Expression Levels:

  • Problem: Low protein yield in expression system.

  • Solution: Optimize codon usage for the expression host, explore different promoters, or try alternative expression systems.

• Inclusion Body Formation:

  • Problem: Protein forms insoluble aggregates.

  • Solution: Lower induction temperature, reduce inducer concentration, co-express with chaperones, or use solubility-enhancing fusion tags.

• Protein Inactivity:

  • Problem: Expressed protein lacks enzymatic activity.

  • Solution: Verify proper folding, ensure appropriate post-translational modifications, check for presence of required cofactors.

• Protein Instability:

  • Problem: Rapid degradation of expressed protein.

  • Solution: Include protease inhibitors during purification, optimize buffer conditions, use stabilizing additives.

• Contamination with Host Proteins:

  • Problem: Difficult to separate recombinant protein from host proteins.

  • Solution: Implement additional purification steps, optimize affinity tag selection and placement.

Systematic optimization of expression conditions and purification protocols is essential for obtaining high-quality recombinant protein.

How can researchers address inconsistent results in enzymatic assays with Recombinant Methanothermobacter marburgensis Putative thymidylate synthase?

Inconsistent assay results can arise from multiple factors:

• Protein Quality Variation:

  • Problem: Batch-to-batch variation in protein activity.

  • Solution: Implement standardized quality control measures, including activity normalization against a reference standard.

• Substrate Quality Issues:

  • Problem: Degraded or impure substrates affecting assay reliability.

  • Solution: Use freshly prepared substrates, verify substrate quality before assays.

• Buffer Composition Effects:

  • Problem: Minor variations in buffer composition affecting enzyme activity.

  • Solution: Prepare master stocks of buffers, carefully control pH and ionic strength.

• Temperature Control:

  • Problem: Inadequate temperature control affecting reaction rates.

  • Solution: Use water-jacketed cuvettes or temperature-controlled plate readers.

• Data Analysis Inconsistencies:

  • Problem: Variations in data processing affecting final results.

  • Solution: Establish standardized data analysis protocols, including appropriate curve fitting and statistical treatments.

Implementing a detailed standard operating procedure (SOP) for all aspects of the enzymatic assay will help minimize variability.

What are the best approaches for analyzing and interpreting contradictory data when studying Methanothermobacter marburgensis Putative thymidylate synthase?

When faced with contradictory data in thymidylate synthase research:

Remember that contradictory data often leads to important discoveries about enzyme mechanisms or experimental artifacts. As emphasized in experimental design literature, understanding the source of counterfactual inference is a central task in research .

How might researchers explore the evolutionary relationships between archaeal thymidylate synthases and their bacterial/eukaryotic counterparts?

Evolutionary studies of thymidylate synthases across domains of life offer rich research opportunities:

• Comprehensive Phylogenetic Analysis:

  • Construct phylogenetic trees using both maximum likelihood and Bayesian approaches.

  • Include representative enzymes from all three domains of life.

  • Analyze patterns of conservation and divergence in catalytic residues.

• Horizontal Gene Transfer Investigation:

  • Examine potential instances of horizontal gene transfer between domains.

  • Compare gene neighborhoods and genomic context across species.

• Structural Comparative Analysis:

  • Compare available crystal structures to identify domain-specific structural features.

  • Map conservation patterns onto structural models to identify functionally important regions.

• Ancestral Sequence Reconstruction:

  • Infer and synthesize ancestral thymidylate synthase sequences.

  • Characterize the properties of reconstructed ancestral enzymes.

• Correlation with Environmental Adaptations:

  • Investigate whether enzyme properties correlate with organism lifestyle.

  • Compare enzymes from extremophiles versus mesophiles.

This research would contribute to understanding the evolution of essential metabolic pathways and could provide insights into the early divergence of the three domains of life.

What novel approaches could be developed for studying the regulation of thymidylate synthase activity in archaeal systems?

Studying regulation in archaeal systems presents unique challenges and opportunities:

These approaches would provide insights into how archaeal cells regulate essential metabolic processes in their unique cellular environments.

How could computational approaches advance our understanding of Methanothermobacter marburgensis Putative thymidylate synthase function and evolution?

Computational methods offer powerful tools for studying enzyme structure, function, and evolution:

• Molecular Dynamics Simulations:

  • Simulate enzyme behavior at different temperatures to understand thermostability.

  • Model substrate binding and catalytic mechanisms at atomic resolution.

  • Investigate the effects of mutations on protein dynamics.

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Model the electronic structure of the active site during catalysis.

  • Calculate energy barriers for different possible reaction mechanisms.

• Machine Learning Applications:

  • Develop models to predict substrate specificity or inhibitor sensitivity.

  • Identify patterns in sequence-function relationships across many homologs.

• Network Analysis:

  • Construct protein-protein interaction networks to understand functional context.

  • Perform metabolic network analysis to identify key control points.

• Computational Docking and Virtual Screening:

  • Screen virtual compound libraries for potential inhibitors.

  • Predict binding modes for substrates and inhibitors.

These computational approaches, integrated with experimental validation, can provide insights that would be difficult to obtain through experimental methods alone.