Recombinant Photobacterium profundum tRNA 2-thiocytidine biosynthesis protein TtcA (ttcA)

What is Photobacterium profundum TtcA and what function does it serve in cellular processes?

TtcA (tRNA 2-thiocytidine biosynthesis protein) from Photobacterium profundum catalyzes the post-transcriptional thiolation of cytosine 32 in specific tRNAs, converting C₃₂ to s²C₃₂ (2-thiocytidine). This modification plays a critical role in maintaining proper tRNA structure and function, contributing to translational accuracy under various environmental conditions. The enzyme employs an iron-sulfur cluster that is essential for its catalytic activity despite catalyzing a non-redox reaction, making it mechanistically distinct from many other tRNA modification enzymes .

P. profundum is a marine bacterium capable of growth at low temperatures and high hydrostatic pressures, with different strains isolated from varying ocean depths displaying remarkable differences in physiological responses to pressure . The function of TtcA must be maintained across these environmental conditions, suggesting potential adaptations in protein structure or regulation.

TtcA operates through an ATP-dependent pathway and requires specific cofactors for activity. In E. coli, studies have shown that TtcA activity depends not only on IscS (a cysteine desulfurase providing sulfur atoms) but also on IscU, presumably required for Fe-S cluster assembly .

How is the TtcA protein structurally characterized with respect to its iron-sulfur cluster?

TtcA protein typically exists as a dimer containing an iron-sulfur cluster. Based on detailed studies of E. coli TtcA (which serves as a model for the P. profundum homolog), the Fe-S cluster in TtcA exists in two forms:

• A [4Fe-4S] form, which is the catalytically active form essential for enzymatic function

• A [2Fe-2S] form, which results from oxygen-induced degradation of the [4Fe-4S] cluster

The Fe-S cluster is coordinated by three conserved cysteine residues: Cys122, Cys125, and Cys222 (numbering based on E. coli TtcA). Site-directed mutagenesis studies have confirmed that these three cysteine residues are essential for cluster formation and enzyme activity, as mutations of any of these cysteines to alanine result in complete loss of s²C₃₂ biosynthesis .

Various spectroscopic techniques have been employed to characterize the Fe-S cluster:

• UV-visible absorption spectroscopy

• Electron Paramagnetic Resonance (EPR) spectroscopy

• Mössbauer spectroscopy

These techniques have confirmed that the [4Fe-4S] form is oxygen-sensitive and prone to decompose into the [2Fe-2S] form and further degraded forms when exposed to oxygen .

What methodologies are recommended for expressing and purifying recombinant P. profundum TtcA?

Based on protocols established for E. coli TtcA, the following methodology can be adapted for the expression and purification of recombinant P. profundum TtcA:

Cloning and Expression Vector Construction:

• Amplify the ttcA gene from P. profundum genomic DNA using PCR with primers containing appropriate restriction sites (such as NdeI and HindIII)

• Clone the gene into an expression vector (e.g., pT7-7) following standard molecular biology techniques

• Consider adding an N-terminal hexahistidine tag for easier purification; functional studies confirm that N-terminal His-tagged TtcA retains full activity in vivo

Protein Expression:

• Transform E. coli BL21(DE3) with the expression plasmid

• Grow cells in appropriate media (LB or marine broth) with antibiotics

• Induce protein expression with IPTG when OD₆₀₀ reaches 0.6-0.8

• Continue growth at lower temperature (15-20°C) to enhance proper folding and Fe-S cluster incorporation

Protein Purification Under Anaerobic Conditions:
Due to the oxygen sensitivity of the [4Fe-4S] cluster, purification should be performed under strict anaerobic conditions:

• Lyse cells using sonication or French press in buffer containing protease inhibitors

• Clarify lysate by centrifugation

• For His-tagged protein, use Ni-NTA affinity chromatography

• Further purify using ion exchange and/or size exclusion chromatography

• Store purified protein under anaerobic conditions with glycerol as a stabilizing agent

Fe-S Cluster Reconstitution:
If the Fe-S cluster is lost during purification, it can be reconstituted:

• Incubate purified apoprotein with ferrous ammonium sulfate and sodium sulfide

• Include DTT as a reducing agent

• Perform the reaction under strict anaerobic conditions

• Remove excess iron and sulfide by desalting or gel filtration

What are the optimal conditions for conducting TtcA activity assays?

Based on studies with E. coli TtcA, the following conditions are recommended for TtcA activity assays:

Required Components:

• Purified TtcA protein containing intact [4Fe-4S] cluster

• tRNA substrate (either total tRNA or specific tRNA species)

• ATP (2-5 mM)

• Magnesium chloride (5-10 mM)

• DTT (1-5 mM)

• Sulfur source (L-cysteine and IscS cysteine desulfurase system)

Buffer Conditions:

• pH: 7.5-8.0

• Buffer: HEPES or Tris-HCl (50-100 mM)

• Salt: NaCl or KCl (50-150 mM)

Reaction Conditions:

• Temperature: 15-25°C (optimal for psychrophilic P. profundum)

• Time: 30-60 minutes

• Anaerobic environment to prevent Fe-S cluster degradation

Detection Methods:

• HPLC analysis of tRNA hydrolysates:

  • Hydrolyze tRNA to nucleosides using nuclease P1 and alkaline phosphatase

  • Analyze using reversed-phase HPLC

  • Detect s²C₃₂ by its characteristic UV absorption and retention time (approximately 10 minutes under standard conditions)

  • Confirm by mass spectrometry (m/z = 260.03 for protonated s²C₃₂)

• In vivo complementation assay:

  • Express TtcA in a ttcA-deficient strain (such as E. coli GRB105)

  • Extract tRNAs and analyze for s²C₃₂ content by HPLC

  • Compare chromatograms with control strains to assess TtcA functionality

How does the presence of the iron-sulfur cluster affect TtcA's function?

The iron-sulfur cluster in TtcA plays a crucial role in enzyme function, despite the fact that the thiolation reaction it catalyzes is not a redox reaction. Research has established several key points:

• Only TtcA containing a [4Fe-4S] cluster is catalytically active, while the [2Fe-2S] form and other degraded forms are inactive .

• The cluster is coordinated by three conserved cysteine residues (Cys122, Cys125, and Cys222), with site-directed mutagenesis confirming that all three are essential for activity .

• The [4Fe-4S] cluster is oxygen-sensitive and can convert to the [2Fe-2S] form upon exposure to oxygen, which explains why anaerobic conditions are critical for maintaining enzyme activity .

The following table summarizes the relationship between cysteine mutations and TtcA activity:

While the exact mechanistic role of the Fe-S cluster remains under investigation, current hypotheses suggest it may:

• Provide structural support for proper protein folding

• Participate in substrate positioning and activation

• Facilitate sulfur transfer from the donor to the cytosine substrate

• Interact with ATP during the catalytic cycle

How do the genomic adaptations of P. profundum strains from different ocean depths affect TtcA expression and function?

P. profundum strains isolated from different ocean depths show remarkable genomic adaptations to their respective environments. The deep-sea piezopsychrophilic strain SS9 and the shallow-water non-piezophilic strain 3TCK display different physiological responses to pressure and other environmental factors .

Genomic Adaptations:
Analysis of sequenced genomes from these strains reveals:

• Variations in gene content between strains

• Specific gene sequences under positive selection

• Differences in gene regulation mechanisms

• Evidence of horizontal gene transfer events that facilitate rapid colonization of new environments

Impact on TtcA Function:
While specific data on ttcA gene expression differences between P. profundum strains is limited, several hypotheses can be formulated:

• Environmental Pressure Effects:
  Deep-sea strains like SS9 may have evolved pressure-responsive regulatory elements in the ttcA promoter region to maintain optimal expression levels under high hydrostatic pressure.

• Protein Structural Adaptations:
  TtcA from deep-sea strains might contain amino acid substitutions that enhance protein stability and function under high pressure and low temperature conditions.

• Fe-S Cluster Stability:
  The stability of the oxygen-sensitive [4Fe-4S] cluster in TtcA might be enhanced in deep-sea strains, as the low oxygen content of deep ocean environments would be less detrimental to cluster integrity.

• tRNA Modification Patterns:
  The extent and pattern of tRNA thiolation might vary between strains as an adaptation to their respective environments, affecting translational fidelity under different growth conditions.

Experimental approaches to investigate these adaptations could include:

• Comparative sequence analysis of ttcA genes and their regulatory regions

• Expression of TtcA from different strains under varying pressure conditions

• Analysis of tRNA modification patterns across strains

• High-pressure enzyme activity assays

What is the proposed mechanism of tRNA 2-thiocytidine biosynthesis catalyzed by TtcA?

Based on biochemical studies of TtcA, a detailed mechanism for tRNA 2-thiocytidine biosynthesis can be proposed:

Proposed Catalytic Mechanism:

• ATP Activation Step:

  • TtcA binds ATP and the target tRNA

  • ATP is hydrolyzed, likely activating the C2 position of cytosine 32

  • This activation may create a good leaving group at the C2 position

• Sulfur Mobilization:

  • The cysteine desulfurase IscS generates a persulfide group

  • This activated sulfur is transferred to TtcA, potentially via the Fe-S cluster

• Fe-S Cluster Involvement:

  • The [4Fe-4S] cluster may coordinate the activated sulfur

  • Alternatively, it may position the cytosine substrate for nucleophilic attack

  • The unique three-cysteine coordination of the cluster leaves one coordination site potentially available for substrate interaction

• Thiolation Reaction:

  • The activated sulfur attacks the C2 position of cytosine 32

  • The leaving group is displaced

  • The 2-thiocytidine (s²C₃₂) is formed

Supporting Evidence:

• ATP Requirement:

  • In vitro assays demonstrate an absolute requirement for ATP

  • ATP and DTT are absolutely required components for enzyme activity

• Fe-S Cluster Essentiality:

  • Only TtcA containing a [4Fe-4S] cluster is active

  • Mutations in the cysteine ligands of the cluster abolish activity

• IscS and IscU Dependency:

  • Genetic studies show that s²C₃₂ biosynthesis depends on both IscS and IscU

  • IscS provides the sulfur, while IscU is required for Fe-S cluster assembly

This mechanism represents TtcA as a unique tRNA-thiolating enzyme that combines features of ATP-dependent enzymes like ThiI and MnmA with the Fe-S cluster dependency of enzymes like MiaB, despite catalyzing a non-redox reaction .

What strategies should be employed to address the oxygen sensitivity of the [4Fe-4S] cluster in experimental design?

The oxygen sensitivity of the [4Fe-4S] cluster in TtcA presents significant challenges for experimental studies. Understanding these challenges and designing experiments accordingly is crucial for obtaining reliable results.

Oxygen Effects on TtcA:

• The [4Fe-4S] cluster degrades to a [2Fe-2S] form upon exposure to oxygen

• Only the [4Fe-4S] form is catalytically active

• Extended oxygen exposure leads to complete loss of the cluster

Experimental Design Strategies:

• Anaerobic Techniques:

  • Use glove boxes or anaerobic chambers for protein purification and assays

  • Degas all buffers and solutions before use

  • Include oxygen scavengers in reaction mixtures

  • Seal reaction vessels to prevent oxygen intrusion

• Protein Expression and Purification:

  • Express protein with iron and sulfur supplements to enhance Fe-S cluster incorporation

  • Purify under strict anaerobic conditions

  • Include stabilizing agents such as DTT and glycerol

• Spectroscopic Monitoring:

  • Use UV-visible spectroscopy to monitor cluster status throughout experiments

  • The [4Fe-4S]² cluster shows characteristic absorption around 400 nm

  • The [2Fe-2S]² cluster shows peaks at ~320, 420, and 460 nm

  • Changes in these spectral features indicate cluster conversion or degradation

• Activity Assays:

  • Perform assays immediately after protein preparation

  • Include controls to assess cluster integrity

  • Consider parallel rather than sequential experiments to minimize time between sample preparation and analysis

• Data Interpretation:

  • Account for potential partial degradation of the Fe-S cluster when analyzing kinetic data

  • Compare enzyme activities only when cluster integrity has been verified

Practical Protocol for Maintaining Cluster Integrity:

• Prepare an anaerobic chamber with N₂/H₂ atmosphere

• Degas all buffers by sparging with N₂ for at least 30 minutes

• Add reducing agents (1-5 mM DTT) to all buffers

• Conduct all protein manipulations in the anaerobic chamber

• Monitor protein by UV-visible spectroscopy before and after each experimental step

• Store protein in sealed containers with oxygen scavengers

What advanced spectroscopic techniques are most informative for characterizing the Fe-S cluster in TtcA?

Comprehensive characterization of the iron-sulfur cluster in TtcA requires a multi-technique approach. The following spectroscopic methods provide complementary information:

• UV-Visible Absorption Spectroscopy:

  • Provides initial evidence of Fe-S cluster presence and type

  • Can monitor cluster degradation over time

  • [4Fe-4S]²⁺ clusters show broad absorption around 400 nm

  • [2Fe-2S]²⁺ clusters show characteristic peaks at ~320, 420, and 460 nm

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Detects paramagnetic species such as reduced [4Fe-4S]¹⁺ clusters

  • The native [4Fe-4S]²⁺ cluster is EPR-silent but can be reduced to the EPR-active state

  • Provides information about the electronic structure and environment of the cluster

  • Typical EPR parameters for [4Fe-4S]¹⁺ clusters include g-values around 1.88-2.06

• Mössbauer Spectroscopy:

  • Provides detailed information about the oxidation and spin states of iron

  • Can distinguish between different types of Fe-S clusters

  • Enables quantification of different iron species in the sample

  • Particularly useful for distinguishing [4Fe-4S] from [2Fe-2S] clusters

• Circular Dichroism (CD) Spectroscopy:

  • Provides information about the environment of the Fe-S cluster

  • Useful for monitoring changes in cluster state under different conditions

• X-ray Absorption Spectroscopy (XAS):

  • Provides information about the local atomic environment around iron atoms

  • Can determine coordination numbers, bond distances, and ligand types

Sample Preparation Considerations:

For EPR analysis:

• Purify TtcA anaerobically

• Prepare samples with 100-200 μM protein in buffer with 10% glycerol

• Reduce a portion of the sample with sodium dithionite

• Transfer samples to EPR tubes and freeze in liquid nitrogen

• Collect EPR spectra at low temperatures (4-20K)

For Mössbauer spectroscopy:

• Grow bacteria in media enriched with ⁵⁷Fe

• Purify protein anaerobically

• Concentrate to 0.5-1 mM protein

• Transfer to Mössbauer sample holders

• Collect spectra at low temperatures (4-80K)

How do mutations in the conserved cysteine residues affect Fe-S cluster assembly and TtcA function?

Studies on E. coli TtcA have provided valuable insights into how mutations in cysteine residues affect the Fe-S cluster and enzyme function. These findings inform research on P. profundum TtcA.

Key Cysteine Residues:
Based on in vivo complementation experiments, three conserved cysteine residues have been identified as essential for TtcA function:

• Cys122

• Cys125

• Cys222

Effects of Cysteine Mutations:

• Impact on Fe-S Cluster Assembly:

  • Mutation of any of the three essential cysteines to alanine results in failure to incorporate the Fe-S cluster

  • Spectroscopic analyses of mutant proteins show absence of the characteristic Fe-S cluster absorption features

• Effects on Enzyme Activity:

  • In vivo complementation experiments in E. coli show that Cys122A, Cys125A, and Cys222A mutants cannot restore s²C₃₂ biosynthesis in ttcA-deficient strains

  • In contrast, mutation of other cysteine residues (Cys146, Cys200, Cys203) has minimal impact on function

• Unique Coordination of the [4Fe-4S] Cluster:

  • The [4Fe-4S] cluster in TtcA appears to be coordinated by only three cysteine residues, unlike the typical four-cysteine coordination seen in many Fe-S proteins

  • This suggests that the fourth coordination site might be occupied by a non-cysteine ligand or be available for substrate binding or catalysis

An experimental strategy for investigating cysteine mutations in P. profundum TtcA would include:

• Identifying conserved cysteine residues through sequence alignment

• Generating site-directed mutants

• Expressing and purifying the mutant proteins

• Characterizing their Fe-S cluster content using spectroscopic methods

• Assessing their ability to catalyze s²C₃₂ formation in vitro and in vivo

What is the evolutionary significance of TtcA in Photobacterium profundum adaptation to different marine environments?

The evolutionary significance of TtcA in P. profundum adaptation to different marine environments encompasses several important aspects:

tRNA Modifications and Environmental Adaptation:
tRNA modifications, including s²C₃₂, play crucial roles in maintaining translational fidelity under various environmental conditions. For P. profundum, which inhabits environments from shallow waters to deep-sea trenches, these modifications may be particularly important for adaptation to:

• Temperature Variation:

  • Deep-sea environments are consistently cold (~2-4°C)

  • Shallow waters experience temperature fluctuations

  • s²C₃₂ modification may stabilize tRNA structure at low temperatures

• Hydrostatic Pressure:

  • Strains like SS9 are adapted to high pressure (piezophilic)

  • tRNA modifications might be important for maintaining proper translation under pressure

  • TtcA activity and Fe-S cluster stability might be differentially regulated in response to pressure

• Oxygen Levels:

  • Oxygen concentration decreases with ocean depth

  • The oxygen-sensitive [4Fe-4S] cluster in TtcA might function better in low-oxygen deep-sea environments

  • This could represent an adaptation that turns a seemingly disadvantageous trait (oxygen sensitivity) into an evolutionary advantage

Comparative Genomics Evidence:
Analysis of P. profundum strains from different depths reveals:

• Conservation of ttcA:

  • The gene is present in both deep-sea and shallow-water strains

  • This suggests a fundamental role in cellular function

• Sequence Variations:

  • Specific substitutions might be under positive selection

  • These could reflect adaptations to different environmental conditions

Horizontal Gene Transfer:
The genome plasticity between Photobacterium bathytypes was demonstrated when strain 3TCK-specific genes for photorepair were introduced to SS9, showing that horizontal gene transfer can provide a mechanism for rapid colonization of new environments . This suggests that similar mechanisms might be involved in the evolution of tRNA modification systems across marine bacterial populations.

How can computational and bioinformatic approaches enhance the study of TtcA homologs across bacterial species?

Bioinformatic approaches provide powerful tools for identifying and characterizing TtcA homologs across bacterial species, including P. profundum. These approaches reveal evolutionary relationships, functional conservation, and potential adaptations.

Sequence-Based Identification and Analysis:

• Homology Searches:

  • Use known TtcA sequences as queries against bacterial genome databases

  • Apply position-specific scoring matrices to identify distant homologs

  • Create comprehensive datasets of TtcA homologs for comparative analysis

• Multiple Sequence Alignment and Conservation Analysis:

  • Identify conserved residues, particularly the three critical cysteines involved in Fe-S cluster coordination

  • Map conservation patterns to functional domains

  • Identify lineage-specific substitutions that might reflect environmental adaptations

Structural Bioinformatics:

• Homology Modeling:

  • Generate structural models of TtcA homologs

  • Analyze the predicted structure of the Fe-S cluster binding site

  • Identify potential functional sites through structural conservation

• Molecular Dynamics Simulations:

  • Simulate TtcA behavior under different environmental conditions (temperature, pressure)

  • Investigate effects of mutations on protein stability and Fe-S cluster coordination

  • Compare dynamics of TtcA from deep-sea vs. shallow-water strains

Machine Learning Approaches:

Similar to the StackTTCA framework described for T-cell antigen identification , machine learning approaches could be applied to TtcA research:

• Feature-Based Classification:

  • Develop models to predict and classify TtcA homologs based on sequence features

  • Distinguish true TtcA enzymes from related proteins

  • Create a probabilistic framework for functional annotation

• Structure-Function Relationship Prediction:

  • Train models to predict the activity or substrate specificity of TtcA homologs

  • Identify key sequence determinants of function

  • Predict the effects of mutations on enzyme activity

Genomic Context Analysis:

• Gene Neighborhood Analysis:

  • Examine genes adjacent to ttcA in different genomes

  • Identify potential functional associations and regulatory elements

  • Detect operons related to tRNA modification systems

• Evolutionary Rate Analysis:

  • Calculate selective pressure (dN/dS ratios) to detect signatures of selection

  • Identify specific sites under positive selection

  • Compare selection patterns between lineages adapted to different environments

A comprehensive bioinformatic pipeline for TtcA analysis would integrate these approaches to provide insights into evolution, function, and environmental adaptation of this important enzyme across bacterial species.