Recombinant Teredinibacter turnerae Thymidylate synthase (thyA)

What is Teredinibacter turnerae and why study its thymidylate synthase?

Teredinibacter turnerae is an intracellular bacterial symbiont that resides in the gills of shipworms, which are wood-eating bivalve mollusks. This bacterium has garnered scientific interest due to its ability to produce cellulolytic enzymes and fix atmospheric nitrogen, potentially contributing to shipworm metabolism in woody environments where nitrogen is limited . T. turnerae was first isolated from shipworms and has been found in various species, including the mangrove shipworm Neoteredo reynei .

The thymidylate synthase (thyA) from T. turnerae merits investigation for several reasons. As an essential enzyme for DNA synthesis, thyA plays a crucial role in the growth and reproduction of this symbiotic bacterium. Research on T. turnerae thyA can provide insights into metabolic adaptations within this unique symbiotic relationship. Additionally, bacterial thyA enzymes can reveal evolutionary patterns and structural differences compared to human thyA, which is a target for anticancer therapeutics. The marine origin of T. turnerae also suggests potential adaptations of its enzymes to function in high-salt environments.

What are the optimal conditions for culturing T. turnerae prior to thyA extraction?

Successful cultivation of T. turnerae requires specific conditions that accommodate its nature as a marine symbiotic bacterium:

Iron availability should be carefully controlled, as T. turnerae produces a siderophore called turnerbactin for iron uptake under iron-limiting conditions . For studies examining iron regulation, the medium can be supplemented with iron chelators like ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDA) . Growth can be monitored by measuring optical density at 600nm, or for cultures with cellulose as carbon source, by visual inspection of cellulose degradation.

What expression systems are recommended for recombinant T. turnerae thyA production?

For recombinant expression of T. turnerae thyA, researchers should consider the following methodological approach:

Vector Construction:
The thyA gene from T. turnerae can be amplified by PCR using primers designed based on the genome sequence. Based on previous work with T. turnerae genes, a successful strategy involves cloning the amplified gene into a T-vector, confirming the sequence, and then subcloning into an expression vector such as pHN33 .

Expression System:
Escherichia coli expression systems (BL21(DE3), Rosetta, or Arctic Express strains) are typically suitable for thyA expression. Given the marine origin of T. turnerae, expression at lower temperatures (16-20°C) may improve proper folding. Consideration of codon optimization may be necessary due to potential differences in codon usage between T. turnerae and E. coli.

Expression Protocol:

• Transform the recombinant plasmid into an appropriate E. coli strain

• Cultivate in LB medium with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

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

• Continue cultivation at reduced temperature (16-20°C) for 16-20 hours

• Harvest cells by centrifugation

Consider including iron in the growth medium, as T. turnerae has iron-dependent regulation systems that might affect protein expression . Additionally, the salt concentration may impact proper folding, as T. turnerae is adapted to marine environments.

What purification strategy should be employed for recombinant T. turnerae thyA?

A comprehensive purification strategy for recombinant T. turnerae thyA typically involves the following steps:

Cell Lysis:

• Resuspend cell pellet in an appropriate lysis buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors)

• Lyse cells by sonication or using a French press

• Clarify the lysate by centrifugation (≥20,000×g for 30 minutes at 4°C)

Affinity Chromatography:
If the recombinant thyA includes a His-tag, a nickel affinity purification approach can be employed:

• Apply clarified lysate to a Ni-NTA or TALON resin column

• Wash with increasing concentrations of imidazole (10-40 mM)

• Elute the target protein with high imidazole concentration (250-300 mM)

Secondary Purification:

• Apply the affinity-purified protein to a size exclusion chromatography column

• Consider ion exchange chromatography if higher purity is required

Quality Control:

• Assess purity by SDS-PAGE (>95% purity is desirable)

• Confirm identity by Western blotting and/or mass spectrometry

• Determine protein concentration using absorbance at 280 nm

• Verify enzymatic activity using a thymidylate synthase activity assay

For storage, purified thyA should be kept in a buffer containing 20-50 mM Tris-HCl pH 7.5, 100-200 mM NaCl, 1-5 mM DTT, and 10-20% glycerol. Aliquots should be flash-frozen in liquid nitrogen and stored at -80°C for long-term use.

How is thymidylate synthase activity measured in laboratory settings?

Several established methods can be employed to measure the enzymatic activity of recombinant T. turnerae thyA:

Spectrophotometric Assay:
This approach monitors the increase in absorbance at 340 nm due to the conversion of 5,10-methylenetetrahydrofolate to dihydrofolate during the catalytic cycle. The reaction mixture typically contains:

• Purified thyA enzyme (0.1-1 μM)

• dUMP (25-100 μM)

• 5,10-methylenetetrahydrofolate (50-200 μM)

• Buffer (50 mM Tris-HCl, pH 7.5)

• Reducing agent (5-10 mM DTT)

• Salt (NaCl, concentration optimized for T. turnerae thyA)

Tritium Release Assay:
This sensitive method uses [5-³H]dUMP as substrate and measures the release of tritium when converted to dTMP. The reaction is typically conducted at 30°C in an appropriate buffer, stopped with charcoal, and the released tritium measured by scintillation counting.

HPLC Analysis:
This approach directly measures the conversion of dUMP to dTMP using HPLC separation. The reaction can be stopped with cold methanol, proteins removed by centrifugation, and the supernatant analyzed by HPLC with UV detection at 260-280 nm.

Coupled Enzyme Assay:
This method couples the production of dihydrofolate to its reduction by dihydrofolate reductase, monitoring NADPH oxidation at 340 nm. This continuous assay allows real-time monitoring of thyA activity.

When analyzing T. turnerae thyA specifically, it's crucial to optimize salt concentration in the assay buffer, as this marine enzyme may have unique salt dependencies for optimal activity.

How does T. turnerae thyA structure compare to other bacterial thymidylate synthases?

While a specific crystal structure for T. turnerae thyA is not yet available in the literature, comparative analysis based on sequence homology and the known structures of related bacterial thymidylate synthases reveals several key features:

T. turnerae thyA likely shares the canonical thymidylate synthase fold, consisting of an eight-stranded β-sheet surrounded by several α-helices. The enzyme is expected to function as a homodimer, with the active site containing conserved residues for substrate binding and catalysis, including a crucial cysteine residue that forms a covalent bond with dUMP during the reaction mechanism.

Given its marine origin, T. turnerae thyA likely displays adaptations to function in high-salt environments. These adaptations may include an altered surface charge distribution, with enhanced negative charge to maintain solubility in high-salt conditions, and specialized salt bridges that stabilize the protein structure.

To experimentally characterize the structural features of T. turnerae thyA, researchers should pursue x-ray crystallography or cryo-electron microscopy studies. Comparative modeling approaches using related bacterial thyA structures can also provide preliminary insights into the three-dimensional architecture of this enzyme.

How might iron availability affect thyA expression and function in T. turnerae?

Iron plays a critical role in T. turnerae metabolism, particularly through the siderophore turnerbactin system. T. turnerae produces turnerbactin, a catechol siderophore required for survival under iron-limiting conditions . While direct evidence for thyA regulation by iron is not currently available, several connections can be inferred:

T. turnerae might have elevated iron requirements due to the need to synthesize iron-rich nitrogenase for nitrogen fixation . This dependence on iron could potentially influence various metabolic pathways, including nucleotide synthesis. In many bacteria, iron limitation affects central metabolism and can alter expression of genes involved in DNA synthesis.

The search results indicate that T. turnerae contains multiple TonB clusters that resemble those found in marine vibrios, with some TonB genes being indispensable for growth under iron-limiting conditions . Interestingly, these TonB genes were further found to be necessary for efficient growth when cellulose was used as the sole carbon source, suggesting connections between iron transport and central metabolism .

To investigate the relationship between iron availability and thyA expression:

• Culture T. turnerae under varying iron conditions:

  • Iron-replete medium (supplemented with ferric ammonium citrate)

  • Iron-limited medium (using iron chelators like EDDA)

  • Iron-limited medium with exogenous turnerbactin addition

• Quantify thyA expression using:

  • Quantitative RT-PCR to measure mRNA levels

  • Western blotting with anti-thyA antibodies to measure protein levels

  • Reporter gene constructs (thyA promoter fused to fluorescent protein)

• Assess thyA enzymatic activity in cell extracts from different iron conditions

These experiments would provide valuable insights into the potential iron-dependent regulation of thyA in this marine symbiotic bacterium.

What role might thyA play in the symbiotic relationship between T. turnerae and shipworms?

The role of thyA in the symbiotic relationship between T. turnerae and shipworms is multifaceted:

DNA Synthesis During Symbiosis:
As the enzyme responsible for thymidylate production, thyA is essential for DNA replication and repair in T. turnerae. Within the context of symbiosis, T. turnerae likely undergoes regulated growth cycles within the shipworm gill tissue, requiring coordinated DNA synthesis. Efficient thyA function would be critical during initial colonization of shipworm gill tissue and maintenance of the established symbiont population.

Metabolic Integration:
T. turnerae produces cellulolytic enzymes and fixes atmospheric nitrogen, contributing to shipworm metabolism in woody environments where nitrogen is restricted . These metabolic contributions require coordinated gene expression and protein synthesis, processes that depend on nucleotide availability and thus indirectly on thyA function.

Potential Regulatory Mechanisms:
The search results indicate that T. turnerae produces turnerbactin, which was detected in the shipworm Lyrodus pedicellatus, suggesting the potential importance of this siderophore in the symbiotic state . The connection between iron acquisition and thyA function might be particularly relevant in the context of symbiosis, as both host and symbiont must coordinate resource allocation.

To investigate the role of thyA in symbiosis, researchers could:

• Compare thyA expression in free-living versus symbiotic T. turnerae using transcriptomic and proteomic approaches

• Create conditional thyA mutants in T. turnerae to assess the impact on colonization efficiency

• Examine co-regulation of thyA with known symbiosis factors

• Analyze thyA sequence conservation across T. turnerae strains isolated from different shipworm species

These approaches would provide insights into how this essential enzyme contributes to the maintenance of this fascinating marine symbiosis.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of T. turnerae thyA?

Site-directed mutagenesis is a powerful approach to investigate the catalytic mechanism of T. turnerae thyA. Key residues for mutation analysis include:

• Catalytic Cysteine: The conserved cysteine that forms a covalent bond with dUMP during catalysis

• Substrate Binding Residues: Arginine residues involved in binding the phosphate group of dUMP

• Folate Binding Residues: Residues that interact with the 5,10-methylenetetrahydrofolate cofactor

• Interface Residues: Amino acids involved in dimer formation

• Marine Adaptation Residues: Surface residues potentially involved in salt tolerance

A comprehensive mutagenesis protocol would involve:

Mutagenesis Protocol:

• Design mutagenic primers containing the desired nucleotide changes

• Perform PCR-based site-directed mutagenesis

• Verify mutations by DNA sequencing

• Express and purify mutant proteins using the established protocol for wild-type

Functional Characterization:
Each mutant should be analyzed for:

• Steady-state kinetic parameters (kcat, Km)

• Structural integrity using circular dichroism or thermal shift assays

• Binding affinity for substrates using isothermal titration calorimetry

• For salt-adaptation studies, activity and stability across salt gradients

The following table presents expected outcomes for key mutations:

Additional approaches could include double mutant cycles to identify cooperative interactions between residues and creation of chimeric enzymes combining regions from T. turnerae thyA with those from other species to identify adaptation-specific domains.

How does salt concentration affect the activity and stability of recombinant T. turnerae thyA?

As T. turnerae is a marine bacterium, salt concentration likely plays a significant role in the activity and stability of its thyA enzyme. The search results indicate that T. turnerae can grow under reduced NaCl concentrations, but certain activities (like cellulose consumption) are clearly reduced under low salt conditions . This suggests that enzymes from this organism, potentially including thyA, are adapted to function optimally in marine salt concentrations.

To investigate salt effects on T. turnerae thyA:

Activity Assays Across Salt Gradient:
Measure thyA enzymatic activity in buffers containing 0-3.5% NaCl, determining both Vmax and Km at different salt concentrations. Test different salt types (NaCl, KCl, MgCl2) to distinguish ionic strength effects from specific ion effects.

Stability Studies:
Monitor thermal denaturation profiles (Tm) using differential scanning fluorimetry across salt concentrations. Perform time-dependent activity assays to measure enzyme half-life at different salt concentrations. Use circular dichroism to assess secondary structure stability in different salt conditions.

Expected salt dependency profiles might include:

The study of salt effects on T. turnerae thyA would not only provide insights into the adaptation of this enzyme to marine environments but could also inform strategies for optimizing the activity and stability of recombinant thyA in various experimental and biotechnological applications.

What challenges might researchers face in crystallizing recombinant T. turnerae thyA?

Crystallizing recombinant T. turnerae thyA for structural studies presents several challenges that researchers should anticipate:

Marine Protein-Specific Challenges:

• Proteins from marine organisms often have adaptations for high-salt environments, which can affect crystallization behavior

• T. turnerae thyA likely requires higher salt concentrations for stability, potentially complicating crystallization conditions

• The surface properties may differ from those of model organisms, affecting crystal contacts

ThyA-Specific Challenges:

• ThyA enzymes undergo conformational changes during catalysis, potentially leading to heterogeneity

• The active enzyme is a homodimer, and ensuring proper dimer formation in crystallization conditions is critical

• Substrate and cofactor binding induces conformational changes that may impact crystal formation

To overcome these challenges, researchers should consider:

Protein Sample Preparation:

• Ensure high purity (>95%) through multiple purification steps

• Verify protein homogeneity using dynamic light scattering

• Test the effect of ligands (dUMP, folate analogs) on protein stability

Crystallization Strategy:

• Screen a wide range of salt conditions, focusing on concentrations reflecting the marine environment

• Explore co-crystallization with substrates, products, or inhibitors

• Consider crystallization of catalytically inactive mutants

• Use microseeding techniques to improve crystal quality

• Employ crystallization additives that mimic the marine environment

The following optimization table provides guidance for crystallization trials:

If crystallization proves particularly challenging, alternative structural approaches such as cryo-electron microscopy could be considered.

How can isotope labeling be used to study the catalytic mechanism of T. turnerae thyA?

Isotope labeling provides powerful tools for investigating the catalytic mechanism of T. turnerae thyA, offering insights into reaction intermediates, transition states, and kinetic isotope effects.

Types of Isotope Labeling for thyA Studies:

• Substrate Labeling:

  • [5-³H]dUMP: Traditional substrate for measuring tritium release during catalysis

  • [¹³C]dUMP: For tracking carbon transfer using NMR or mass spectrometry

  • [¹⁵N]dUMP: For monitoring nitrogen involvement in the reaction

• Cofactor Labeling:

  • [¹³C]5,10-methylenetetrahydrofolate: For tracking methyl transfer

  • Deuterated folate derivatives: For measuring kinetic isotope effects

• Enzyme Labeling:

  • [¹³C/¹⁵N]thyA: Uniform labeling for NMR studies

  • Selective isotope labeling of specific amino acids (e.g., [¹³C]Cys for active site studies)

Experimental Design for Mechanism Elucidation:

A specific protocol for isotope labeling studies with T. turnerae thyA would include:

• Express recombinant T. turnerae thyA in minimal media with specific isotope-labeled precursors

• Purify the labeled enzyme using established protocols

• Perform steady-state kinetics with isotope-labeled substrates

• Calculate kinetic isotope effects from the ratio of rates

• Use NMR spectroscopy to monitor chemical shift changes during the reaction

• Correlate experimental findings with quantum mechanical calculations

These approaches would provide valuable mechanistic insights, potentially revealing unique aspects of the catalytic mechanism of thyA from this marine symbiotic bacterium.