Recombinant Geobacter sulfurreducens Thymidylate synthase thyX (thyX)

What is ThyX and how does it differ from classical thymidylate synthase (ThyA)?

ThyX is an alternative thymidylate synthase that catalyzes the transfer of a methyl group from methylenetetrahydrofolate (CH₂H₄-folate) to dUMP, forming dTMP, which is essential for DNA synthesis . Unlike the classical ThyA enzyme found in most eukaryotes, ThyX has a completely different structure and enzymatic mechanism .

Key differences include:

ThyX represents a distinct evolutionary solution to thymidylate synthesis, making it particularly interesting for comparative enzymology studies and antimicrobial development .

What are the key characteristics of Geobacter sulfurreducens relevant to recombinant ThyX research?

Geobacter sulfurreducens possesses several distinctive characteristics that influence recombinant protein expression and function:

• It is a gram-negative, metal- and sulfur-reducing proteobacterium with a unique metabolism .

• G. sulfurreducens has an unusually high iron content (2 ± 0.2 μg/g dry weight) due to its extensive cytochrome network .

• The organism has high lipid content (32 ± 0.5% dry weight/dry weight), which reflects its specialized membrane composition .

• Though traditionally classified as a strict anaerobe, G. sulfurreducens is now recognized as a facultative microaerobe that can utilize oxygen as a terminal electron acceptor when concentrations are not excessive .

• G. sulfurreducens exhibits electroactivity, able to transfer electrons to metallic minerals and electrodes, making it a model "electricigen" .

These characteristics may affect the expression, folding, and activity of recombinant ThyX, particularly regarding cofactor incorporation and redox properties .

Why is recombinant G. sulfurreducens ThyX of interest to researchers?

Recombinant G. sulfurreducens ThyX is of significant interest for several compelling reasons:

• Antimicrobial target potential: ThyX is present in many pathogenic bacteria but absent in humans, making it a promising target for novel antibiotics with potentially fewer side effects .

• Unique enzymatic mechanism: G. sulfurreducens ThyX represents a distinct class of thymidylate synthases with a flavin-dependent mechanism that differs fundamentally from the classical ThyA enzyme .

• Relationship to electroactive metabolism: Understanding ThyX in G. sulfurreducens may provide insights into how this essential DNA synthesis pathway functions in an organism with unique electron transfer capabilities .

• Structural flexibility insights: ThyX enzymes demonstrate remarkable active site flexibility to accommodate multiple substrates, making them interesting models for studying enzyme dynamics and substrate recognition .

• Evolutionary significance: Study of bacterial enzymes that perform essential functions through alternative mechanisms provides insights into evolutionary diversity and adaptation .

The recombinant expression systems for G. sulfurreducens ThyX enable detailed biochemical and structural studies that would be difficult to perform with the native enzyme in its original host .

How can recombinant G. sulfurreducens ThyX be expressed and purified for research purposes?

Expression and purification of recombinant G. sulfurreducens ThyX can be accomplished through a standardized methodology similar to approaches used for other ThyX proteins:

• Cloning strategy:

  • Amplify the thyX gene from G. sulfurreducens genomic DNA using PCR with specific primers containing appropriate restriction sites

  • Clone the amplified gene into an expression vector (e.g., pQE80L) that incorporates an N-terminal histidine tag for purification

  • Verify the construct by DNA sequencing

• Expression conditions:

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

  • Induce protein expression with IPTG (typically 0.5-1 mM) when cultures reach mid-log phase

  • Optimize expression by testing different temperatures (18-37°C), induction times (3-24 hours), and media compositions

• Purification protocol:

  • Harvest cells by centrifugation and lyse using sonication or French press

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

  • Purify using nickel affinity chromatography with imidazole gradient elution

  • Further purify by size exclusion chromatography if needed

  • Verify purity by SDS-PAGE and protein identity by Western blotting with ThyX-specific antibodies

• Quality assessment:

  • Measure protein concentration using Bradford or BCA assay

  • Verify flavin incorporation by measuring absorbance at 450 nm (characteristic of FAD)

  • Assess enzymatic activity using the standard thymidylate synthase assay

The purified recombinant protein typically appears yellow due to bound flavin cofactor, which is essential for its catalytic activity .

What methods are used to measure ThyX enzymatic activity and what are the optimal assay conditions?

Several complementary methods can be used to measure ThyX enzymatic activity:

• Tritium release assay:

  • This classic method measures the release of tritium from [5-³H]dUMP during the methylation reaction

  • Reaction mixture typically contains:

    • 50-100 mM buffer (HEPES or Tris, pH 7.5-8.0)

    • 5-20 μM [5-³H]dUMP

    • 0.2-1 mM CH₂H₄-folate

    • 0.1-0.5 mM NADPH

    • 0.5-5 μg purified ThyX enzyme

  • After incubation (typically 10-30 minutes at 37°C), unreacted [5-³H]dUMP is removed by activated charcoal

  • Released ³H₂O is quantified by liquid scintillation counting

• Spectrophotometric NADPH oxidation assay:

  • Monitors the decrease in absorbance at 340 nm as NADPH is oxidized during the reaction

  • Useful for continuous monitoring of activity and initial rate measurements

  • The reaction composition is similar to the tritium release assay but uses non-radioactive dUMP

• HPLC-based product detection:

  • Directly measures dTMP formation using HPLC separation

  • Provides absolute quantification of product formation

  • Can be coupled with mass spectrometry for additional verification

Optimal assay conditions for G. sulfurreducens ThyX typically include:

• Temperature: 30-37°C (reflecting the mesophilic nature of G. sulfurreducens)

• pH: 7.5-8.0

• Divalent cations: 5-10 mM Mg²⁺

• Reducing environment: 1-5 mM DTT or β-mercaptoethanol

One unit of enzyme activity corresponds to the production of 1 nmol of dTMP synthesized per minute under optimum assay conditions .

How can functional complementation assays be used to verify ThyX activity?

Functional complementation assays provide a powerful way to verify ThyX activity in a biological context:

• Experimental approach:

  • Transform a thymidylate synthase-deficient strain of E. coli (e.g., thyA⁻ mutant) with the recombinant plasmid expressing G. sulfurreducens ThyX

  • Plate transformed cells on minimal medium lacking thymidine

  • Observe growth rescue, which indicates functional ThyX activity

  • Include appropriate controls: empty vector (negative control) and E. coli thyA (positive control)

• Advantages of this method:

  • Demonstrates biological relevance of the enzyme beyond in vitro activity

  • Confirms that the recombinant enzyme can function in a heterologous cellular environment

  • Provides a robust qualitative assessment of activity

• Quantitative analysis:

  • Growth rates in liquid culture can be measured to quantitatively assess complementation efficiency

  • Different ThyX variants or mutants can be compared based on their ability to support growth

  • Complementation under various stress conditions can provide insights into enzyme robustness

• Limitations and considerations:

  • Successful complementation depends on proper expression, folding, and cofactor incorporation

  • Growth may be slower than with the native ThyA due to mechanistic differences

  • The assay only works if the host strain cannot bypass the need for thymidylate synthase

This approach has been successfully used to verify activity of recombinant ThyX proteins from various bacterial sources, including Parachlamydia UWE25 ThyX .

What is known about the active site flexibility of ThyX and how does it impact substrate binding?

The active site of ThyX exhibits remarkable conformational flexibility that is critical for its function:

• Experimental evidence of flexibility:

  • Femtosecond time-resolved fluorescence spectroscopy reveals that ThyX active site dynamics span three orders of magnitude in timescale

  • Multiple angstrom-scale cofactor-residue displacements occur on the picosecond timescale at physiological temperatures

  • Molecular dynamics simulations support these experimental findings

• Substrate-induced conformational changes:

  • dUMP binding abolishes this flexibility and stabilizes the active site

  • dUMP closely interacts with the flavin cofactor and efficiently quenches its fluorescence

  • This stabilization may facilitate binding of subsequent substrates (NADPH, MTHF)

• Mechanistic implications:

  • ThyX follows a "dynamic selected-fit mechanism" where binding of the first substrate (dUMP) stabilizes the enzyme in a configuration favorable for interaction with subsequent substrates

  • This multi-substrate binding capability is essential for ThyX function since it must accommodate dUMP, MTHF, and NADPH

• Structural considerations:

  • In substrate-free structures, the flavin group and its environment appear disordered

  • Multiple flavin configurations have been observed in folate-bound forms

  • dUMP binds in close interaction with the flavin group, displacing a nearby tyrosine residue

This inherent flexibility likely explains how ThyX can efficiently coordinate the binding and chemistry of multiple substrates in its catalytic cycle, making it an intriguing model for studying enzyme dynamics in multi-substrate reactions .

How does SigB regulation affect ThyX expression in bacteria, and does this apply to G. sulfurreducens?

SigB regulation of ThyX has been primarily characterized in Corynebacterium glutamicum, with potential implications for other bacteria including G. sulfurreducens:

• Evidence for SigB regulation:

  • In C. glutamicum, ThyX expression is regulated by the alternative sigma factor SigB

  • In a ΔsigB strain, ThyX levels are significantly diminished compared to wild-type

  • Complementation of the sigB gene restores ThyX expression to wild-type levels

• Growth phase-dependent expression:

  • ThyA levels decrease gradually during stationary phase

  • ThyX levels are maintained steadily throughout stationary phase

  • This suggests different roles for the two thymidylate synthases during various growth phases

• Functional implications:

  • SigB is necessary for maintaining ThyX levels during transition into stationary phase

  • Growth of ΔsigB strains depends on coupling activity of dihydrofolate reductase (DHFR) with ThyA

  • This dependency makes ΔsigB strains sensitive to DHFR inhibitors like WR99210-HCl

• Potential relevance to G. sulfurreducens:

  • While direct evidence for SigB regulation in G. sulfurreducens ThyX is not provided in the search results

  • G. sulfurreducens undergoes significant transcriptional changes in response to environmental conditions such as oxygen levels

  • It's possible that G. sulfurreducens employs similar regulatory mechanisms to modulate ThyX expression under stress conditions

This regulatory relationship suggests that ThyX may play a particularly important role during stationary phase or stress conditions, which could be relevant when studying recombinant ThyX expression or designing inhibitors targeting this enzyme .

How does oxygen tolerance in G. sulfurreducens affect ThyX activity and experimental design considerations?

G. sulfurreducens was originally considered a strict anaerobe but is now recognized as a facultative microaerobe, which has important implications for ThyX research:

• Oxygen tolerance and utilization:

  • G. sulfurreducens can not only tolerate oxygen but can use it as a terminal electron acceptor

  • Growth with oxygen is possible if the maximum specific oxygen uptake rate (sOUR) of 95 mg O₂ g CDW⁻¹ h⁻¹ is not exceeded

  • At higher oxygen concentrations, growth is completely inhibited

• Oxygen response strategies:

  • Transcriptome analysis reveals three distinct survival strategies depending on oxygen concentration:

    • At low oxygen levels: G. sulfurreducens attempts to escape the microaerobic area

    • At intermediate levels: Cells focus on rapid and complete oxygen reduction coupled to growth

    • At high levels: Cells form protective layers when complete reduction becomes impossible

• Implications for ThyX activity:

  • ThyX is a flavoenzyme that participates in redox chemistry, which could be affected by oxygen levels

  • The extensive cytochrome network in G. sulfurreducens that responds to oxygen may interact with ThyX function

  • Oxygen tension could affect the redox state of the FAD cofactor in ThyX

• Experimental design considerations:

  • Purification of recombinant ThyX may require controlled oxygen conditions to maintain proper cofactor redox state

  • Activity assays should consider potential oxygen effects on ThyX function

  • When expressing recombinant G. sulfurreducens ThyX, the oxygen tolerance of the expression host becomes relevant

  • For functional studies, researchers should clearly define and control oxygen conditions

The oxygen adaptability of G. sulfurreducens adds a layer of complexity to ThyX research and may provide opportunities to study how this enzyme functions under varying redox conditions .

How can computational approaches be used to identify ThyX inhibitors as potential antimicrobial agents?

Computational methods have proven valuable for identifying ThyX inhibitors as potential antimicrobial agents:

• Bayesian modeling approach:

  • Training sets can be developed by testing compounds (e.g., 94 molecules) against ThyX at a standardized concentration (e.g., 100 μM)

  • Molecules with >70% inhibition can be classified as actives to generate a Bayesian model

  • The resulting model can predict inhibitory activity of untested compounds

  • Successful models achieve ROC scores of 0.78-0.80 after cross-validation, with sensitivity, specificity, and concordance values >0.90

• Structure-activity insights:

  • Quinones and naphthoquinones (NQs) have been identified as important structural features for ThyX inhibition

  • Compounds with amines and sulfonamides show reduced inhibitory activity

  • These structure-activity relationships guide future inhibitor design

• Validation of computational predictions:

  • When testing 14 compounds predicted by Bayesian models, 50% of those predicted as actives exhibited >70% inhibition of Mycobacterium tuberculosis ThyX in vitro

  • This demonstrates the robustness and utility of computational models in accelerating inhibitor discovery

• Application to G. sulfurreducens ThyX:

  • Similar computational approaches could be applied to G. sulfurreducens ThyX

  • Models would need to be retrained with experimental data specific to G. sulfurreducens ThyX

  • Potential inhibitors identified through this approach would require experimental validation

• Advantages of computational methods:

  • Rapid screening of large compound libraries

  • Cost-effective prioritization of compounds for experimental testing

  • Ability to share models with other researchers through platforms like CDD Models

This target-based machine learning approach represents an efficient strategy for identifying enzyme inhibitors that could be applied to G. sulfurreducens ThyX as well as ThyX enzymes from other bacteria .

What is the essential function of ThyX in bacteria that also possess ThyA, and how does this impact antimicrobial targeting strategies?

The presence of both ThyA and ThyX in some bacteria, including mycobacteria, raises intriguing questions about their respective functions:

• Evidence for ThyX essentiality:

  • In Mycobacterium tuberculosis, ThyX is essential even in the presence of ThyA

  • ThyA can be successfully deleted from the M. tuberculosis genome, resulting in only an in vitro growth defect

  • The requirement for ThyX despite the presence of ThyA suggests it performs an essential function beyond dTMP synthesis

• Alternative essential functions of ThyX:

  • While both ThyA and ThyX catalyze the methylation of dUMP to form dTMP, ThyX may have additional roles

  • The flavin-dependent mechanism of ThyX may be important for maintaining redox balance

  • ThyX might play a role in stress responses or stationary phase survival, as suggested by its SigB-regulated expression

• Implications for antimicrobial targeting:

  • The essentiality of ThyX makes it a promising target for novel antimicrobials

  • Inhibitors specific to ThyX would not affect human ThyA, potentially reducing side effects

  • The unique active site flexibility and flavin-dependent mechanism of ThyX provide opportunities for selective inhibition

  • Understanding the essential non-dTMP synthase function of ThyX could reveal additional targeting strategies

• Growth phase considerations:

  • ThyA levels decrease during stationary phase while ThyX levels remain steady

  • This suggests ThyX may be particularly important during stationary phase or stress conditions

  • Antimicrobial strategies targeting ThyX might be especially effective against persistent bacterial infections

This complex relationship between ThyA and ThyX highlights the importance of understanding the specific roles of ThyX in different bacteria, including G. sulfurreducens, for effective antimicrobial development .

How can ultrafast time-resolved fluorescence spectroscopy be applied to study conformational dynamics of ThyX?

Ultrafast time-resolved fluorescence spectroscopy provides valuable insights into ThyX conformational dynamics:

• Methodological approach:

  • The technique exploits the natural fluorescence of the FAD cofactor in ThyX

  • Electron transfer from a neighboring tyrosine residue to excited FAD quenches fluorescence

  • The dynamics of this quenching process serve as a sensitive probe of the active site configuration

  • Measurements across different temperatures provide additional insights into conformational energy landscapes

• Key findings for ThyX:

  • Fluorescence decay spans three orders of magnitude, demonstrating a wide range of conformations

  • Multiple angstrom cofactor-residue displacements occur on the picosecond timescale at physiological temperatures

  • dUMP binding abolishes this flexibility and stabilizes the active site

  • These results indicate a "dynamic selected-fit mechanism" where substrate binding progressively stabilizes the enzyme

• Technical requirements:

  • Femtosecond laser systems capable of generating ultrashort pulses

  • Time-correlated single photon counting or streak camera detection systems

  • Temperature-controlled sample chambers

  • Analysis software for complex multiexponential decay modeling

• Application to G. sulfurreducens ThyX:

  • This technique could reveal whether G. sulfurreducens ThyX exhibits similar conformational dynamics

  • The electroactive nature of G. sulfurreducens might influence the redox properties and dynamics of the FAD cofactor

  • Comparative studies could highlight any unique features of G. sulfurreducens ThyX related to its specialized metabolism

• Complementary approaches:

  • Molecular dynamics simulations can provide theoretical support for experimental findings

  • Single-molecule FRET could provide additional insights into conformational distributions

  • Hydrogen-deuterium exchange mass spectrometry could map flexible regions

This approach enables real-time monitoring of ThyX conformational evolution on intrinsic picosecond timescales, providing unique insights impossible to obtain through static structural methods .

What are the challenges and best practices for expressing recombinant ThyX from anaerobic or microaerobic bacteria in E. coli?

Expressing recombinant ThyX from anaerobic or microaerobic bacteria like G. sulfurreducens in E. coli presents several challenges:

• Oxygen sensitivity considerations:

  • While G. sulfurreducens can tolerate oxygen under certain conditions, its proteins may have evolved in primarily anaerobic environments

  • The flavin cofactor in ThyX is susceptible to oxidation, potentially affecting activity

  • Expression and purification protocols may need to incorporate anaerobic techniques or reducing agents

• Cofactor incorporation:

  • Proper incorporation of the FAD cofactor is essential for ThyX activity

  • E. coli may not provide sufficient FAD under standard growth conditions

  • Supplementation with riboflavin or FAD in the growth medium may enhance cofactor incorporation

  • Verification of flavin content by absorbance spectroscopy (peak at ~450 nm) is crucial

• Codon optimization:

  • G. sulfurreducens has a different codon usage bias than E. coli

  • Codon optimization of the thyX gene for E. coli expression can improve protein yields

  • Alternatively, using E. coli strains with additional tRNA genes (e.g., Rosetta strains) can address rare codon issues

• Solubility enhancement strategies:

  • Fusion tags beyond His-tags (e.g., MBP, SUMO) may improve solubility

  • Expression at lower temperatures (16-20°C) often improves folding and solubility

  • Co-expression with chaperones may assist proper folding

• Verified expression approach:

  • Clone the thyX gene into a vector with an N-terminal His-tag for purification

  • Transform into an appropriate E. coli expression strain

  • Express at lower temperature (18-25°C) after induction

  • Confirm yellow color of purified protein, indicating flavin incorporation

  • Verify activity through complementation of a thyA-deficient E. coli strain

• Functional validation:

  • Complementation of thyA-deficient E. coli provides robust validation of function

  • In vitro activity assays should include proper controls and cofactors

  • Comparison with well-characterized ThyX enzymes helps validate the expression system

These considerations help ensure that recombinant G. sulfurreducens ThyX retains its native structure and function when expressed in heterologous systems .

How might the electroactive properties of G. sulfurreducens influence ThyX function, and what experimental approaches could explore this relationship?

The unique electroactive properties of G. sulfurreducens may have intriguing implications for ThyX function:

• Potential relationships between electroactivity and ThyX:

  • G. sulfurreducens has an extensive network of cytochromes for extracellular electron transfer

  • ThyX depends on redox chemistry involving its FAD cofactor

  • The electron transport systems in G. sulfurreducens could potentially interact with or influence ThyX redox cycling

  • The high iron content (2 ± 0.2 μg/g dry weight) in G. sulfurreducens cells may create a unique redox environment for ThyX

• Experimental approaches to explore this relationship:

  • Comparative enzyme kinetics: Measure ThyX activity from G. sulfurreducens versus other bacteria under various redox conditions

  • Electrode-coupled enzyme assays: Develop systems where ThyX activity can be measured while controlling redox potential via electrodes

  • Protein-protein interaction studies: Investigate whether ThyX interacts with components of the electron transport chain

  • In vivo studies: Examine ThyX expression and activity in G. sulfurreducens grown under electrode-respiring versus fumarate-respiring conditions

• Technical considerations:

  • Bioelectrochemical systems can provide controlled redox environments

  • Spectroelectrochemistry could monitor ThyX cofactor redox states under applied potentials

  • Anaerobic chambers would be necessary for many experiments

  • Genetic systems in G. sulfurreducens allow for creation of reporter constructs

• Potential significance:

  • Could reveal novel regulatory mechanisms for ThyX based on cellular redox state

  • Might identify unique adaptations of ThyX in electroactive bacteria

  • Could inform the development of bioelectronic systems incorporating ThyX activity

This emerging research direction could provide insights into how essential cellular processes like DNA synthesis are integrated with the unique electron transfer capabilities of G. sulfurreducens .

What role might ThyX play in the survival strategies of G. sulfurreducens under varying oxygen conditions?

G. sulfurreducens exhibits sophisticated survival strategies under different oxygen conditions, which may involve ThyX:

• G. sulfurreducens oxygen response strategies:

  • At low oxygen levels: Cells attempt to escape the microaerobic area

  • At intermediate levels: Cells focus on rapid oxygen reduction coupled to growth

  • At high levels: Cells form protective layers when complete reduction is impossible

• Potential roles of ThyX in oxygen response:

  • Redox balancing: ThyX uses NADPH and involves flavin redox cycling, potentially contributing to cellular redox homeostasis under oxidative stress

  • DNA synthesis under stress: Maintaining ThyX activity could be crucial for DNA replication and repair under oxidative stress

  • Alternative functions: As suggested for M. tuberculosis, ThyX might have essential functions beyond dTMP synthesis that become important under stress conditions

• Experimental approaches to investigate:

  • Transcriptome analysis: Compare thyX expression levels under anaerobic versus microaerobic conditions

  • Conditional knockdown: Create thyX conditional mutants and test survival under different oxygen tensions

  • Biochemical characterization: Test whether oxygen affects ThyX activity in vitro

  • Metabolomic analysis: Measure dTMP and related metabolite levels under varying oxygen conditions

• Technical considerations:

  • Precise control of oxygen concentration is critical (below 95 mg O₂ g CDW⁻¹ h⁻¹)

  • Continuous monitoring of oxygen levels during experiments

  • Consider the role of the menaquinol oxidase, which is likely responsible for oxygen reduction in G. sulfurreducens

Understanding the role of ThyX in oxygen response could provide insights into how G. sulfurreducens maintains essential cellular processes during transitions between anaerobic and microaerobic environments, potentially revealing novel regulatory mechanisms for this essential enzyme .

How could inhibitor development for ThyX incorporate the unique aspects of G. sulfurreducens metabolism?

Developing inhibitors for G. sulfurreducens ThyX that leverage the organism's unique metabolism presents an innovative research direction:

• Exploiting redox sensitivity:

  • G. sulfurreducens has extensive electron transfer capabilities and cytochrome networks

  • Inhibitors that become activated under specific redox conditions might selectively target G. sulfurreducens ThyX

  • Redox-cycling compounds could potentially interfere with both ThyX function and the electron transfer systems

• Membrane-targeting strategies:

  • G. sulfurreducens has unusually high lipid content (32 ± 0.5% dry weight/dry weight)

  • Inhibitors with appropriate lipophilicity might accumulate in G. sulfurreducens membranes

  • Conjugating ThyX inhibitors to membrane-targeting moieties could enhance selectivity

• Iron-dependent approaches:

  • The high iron content of G. sulfurreducens (2 ± 0.2 μg/g dry weight) suggests iron-dependent processes are important

  • Inhibitors that chelate iron or interfere with iron-dependent pathways might create synergistic effects with ThyX inhibition

  • Iron-binding moieties could potentially be incorporated into ThyX inhibitor scaffolds

• Oxygen-sensitive inhibitors:

  • G. sulfurreducens can grow under microaerobic conditions but has specific oxygen tolerance thresholds

  • Compounds that become more potent ThyX inhibitors upon oxygen exposure might be effective

  • This approach could target G. sulfurreducens specifically in microaerobic niches

• Experimental approaches:

  • Apply Bayesian modeling approaches similar to those used for M. tuberculosis ThyX

  • Test inhibitor efficacy under various growth conditions (anaerobic, microaerobic, with different electron acceptors)

  • Evaluate synergy between ThyX inhibitors and compounds targeting electron transport chains

  • Incorporate structure-based design using homology models of G. sulfurreducens ThyX

This approach integrates knowledge of G. sulfurreducens' unique cellular composition and metabolism with ThyX inhibitor design, potentially leading to more selective and effective compounds for basic research or biotechnological applications .

What are the most promising future research directions for recombinant G. sulfurreducens ThyX?

Several promising research directions emerge for recombinant G. sulfurreducens ThyX:

• Structural and functional characterization:

  • Determining the high-resolution structure of G. sulfurreducens ThyX

  • Applying ultrafast time-resolved fluorescence spectroscopy to study its conformational dynamics

  • Investigating potential unique features related to the electroactive nature of G. sulfurreducens

• Integration with electron transfer systems:

  • Exploring potential interactions between ThyX and components of G. sulfurreducens' extensive cytochrome network

  • Developing electrode-coupled systems to study ThyX function under controlled redox conditions

  • Investigating how ThyX activity responds to different electron acceptors (fumarate, Fe(III), electrodes)

• Inhibitor development:

  • Applying machine learning approaches to identify selective inhibitors

  • Exploring compounds that leverage G. sulfurreducens' unique metabolism

  • Testing inhibitor efficacy under various environmental conditions

• Synthetic biology applications:

  • Engineering ThyX for improved catalytic efficiency or novel substrate specificity

  • Incorporating ThyX into synthetic pathways for nucleotide production

  • Exploring the potential of ThyX in bioelectronic systems

• Essential function determination:

  • Investigating whether G. sulfurreducens ThyX has essential functions beyond dTMP synthesis

  • Comparing thyA and thyX expression under various growth conditions and stresses

  • Creating conditional mutants to study differential roles of ThyA and ThyX