Recombinant Dehalococcoides sp. Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase and what is its primary function?

Queuine tRNA-ribosyltransferase (TGT) is the key enzyme in the queuosine pathway, responsible for the exchange of guanine at position 34 of target tRNAs with the precursor base preQ1. This exchange is a critical step in the incorporation of queuosine (Q), a 7-deaza-guanosine derivative present in tRNA anticodons . The enzyme catalyzes this base exchange reaction as part of the tRNA modification machinery that ensures proper protein synthesis and translation fidelity in various organisms, including Dehalococcoides species .

How does the structure of Dehalococcoides sp. TGT compare to eukaryotic and other bacterial TGTs?

The Dehalococcoides sp. TGT functions as part of the bacterial tRNA modification machinery. Unlike eukaryotic TGTs which exist as heterodimers (as seen in human TGT composed of hQTRT1 and hQTRTD1 subunits), bacterial TGTs typically function as homodimers . The Dehalococcoides species contains genes related to the queuosine synthesis pathway, including a functional TGT enzyme that has been shown to participate in the tRNA modification process . The bacterial TGT enzyme shares the core catalytic mechanism with eukaryotic counterparts but differs in quaternary structure and specific substrate recognition patterns.

What tRNA substrates are recognized by Dehalococcoides sp. TGT?

Dehalococcoides sp. TGT, like other bacterial TGTs, primarily recognizes tRNAs with GUN anticodons, specifically tRNAs for tyrosine (Tyr), histidine (His), asparagine (Asn), and aspartic acid (Asp) . These target tRNAs contain guanine at position 34 (the wobble position of the anticodon), which is replaced with preQ1 by the TGT enzyme. This specificity for certain tRNA isoacceptors is consistent across most bacterial species, though the efficiency of modification may vary based on structural elements within both the enzyme and the tRNA substrates.

What expression systems are most effective for producing recombinant Dehalococcoides sp. TGT?

For recombinant expression of Dehalococcoides sp. TGT, Escherichia coli-based expression systems have proven effective. Specifically, approaches similar to those used for other bacterial TGTs can be employed, such as using pET-based vectors with His-tag fusions for facile purification . When expressing recombinant TGT from Dehalococcoides ethenogenes 195, researchers have successfully used E. coli expression systems with appropriate codon optimization to account for the different GC content of Dehalococcoides species . Expression conditions typically involve induction at lower temperatures (16-25°C) to enhance proper folding and reduce inclusion body formation, with IPTG concentrations of 0.1-0.5 mM being optimal for controlled expression.

What purification strategy yields the highest activity for recombinant Dehalococcoides sp. TGT?

A multi-step purification approach is recommended for obtaining high-activity recombinant Dehalococcoides sp. TGT:

• Initial capture using Ni²⁺ affinity chromatography for His-tagged enzyme

• Ion exchange chromatography (typically anion exchange) to remove contaminants

• Size exclusion chromatography for final polishing and buffer exchange

Maintaining reducing conditions throughout purification is critical, as conserved cysteines in the protein may be involved in coordinating Fe/S centers similar to what has been observed in related enzymes . Purification buffers typically contain 5-10% glycerol and reducing agents such as DTT or β-mercaptoethanol to maintain enzyme stability. The purified enzyme should be stored in buffer containing stabilizing agents at -80°C to preserve activity.

How can I assess the purity and integrity of recombinant Dehalococcoides sp. TGT?

Multiple analytical methods should be employed to assess the purity and integrity of recombinant Dehalococcoides sp. TGT:

• SDS-PAGE to verify protein size and purity (>95% homogeneity is desirable)

• Western blotting using anti-His antibodies if a His-tagged construct is used

• Size exclusion chromatography to confirm proper oligomeric state

• Mass spectrometry to verify the correct molecular weight and identify potential post-translational modifications

• Circular dichroism spectroscopy to assess proper folding

• Activity assays using model tRNA substrates to confirm functional integrity

For definitive structural characterization, techniques such as X-ray crystallography or cryo-electron microscopy could be employed, though these approaches are more resource-intensive.

What are the optimal assay conditions for measuring Dehalococcoides sp. TGT activity?

Optimal conditions for assaying Dehalococcoides sp. TGT activity typically include:

Activity is typically measured by monitoring the incorporation of radiolabeled bases into tRNA substrates or through more modern approaches using fluorescently labeled tRNAs coupled with electrophoretic separation or HPLC analysis .

How does the catalytic efficiency of Dehalococcoides sp. TGT compare with TGTs from other organisms?

The catalytic efficiency (k₍cat₎/K₍M₎) of TGT enzymes can vary significantly between species. While specific kinetic parameters for Dehalococcoides sp. TGT have not been extensively reported in the provided search results, comparative studies with other bacterial TGTs suggest similar enzymatic mechanisms. For example, the human TGT (hQTRT1- hQTRTD1) using human tRNA^Tyr^ and guanine shows catalytic efficiency comparable to that of E. coli TGT .

Bacterial TGTs generally exhibit K₍M₎ values in the low micromolar range for tRNA substrates and k₍cat₎ values of 0.1-1.0 min⁻¹. The Dehalococcoides sp. TGT likely falls within this range, though specific determination requires direct experimental measurement. Functional complementation experiments suggest that the Dehalococcoides ethenogenes 195 enzyme possesses sufficient catalytic activity to functionally replace similar enzymes in heterologous systems .

What is the role of metal cofactors in Dehalococcoides sp. TGT activity?

Metal cofactors play crucial roles in the structure and function of Dehalococcoides sp. TGT and related enzymes. While the search results don't provide specific details about metal requirements for Dehalococcoides sp. TGT, related enzymes in the queuosine modification pathway contain conserved cysteines involved in coordinating Fe/S centers . For the TGT enzyme specifically, divalent metal ions (particularly Mg²⁺) are essential cofactors that stabilize the enzyme-substrate complex and facilitate the base exchange reaction.

How can recombinant Dehalococcoides sp. TGT be used for studying tRNA modification pathways?

Recombinant Dehalococcoides sp. TGT serves as a valuable tool for studying tRNA modification pathways through multiple approaches:

• In vitro reconstitution systems: Purified recombinant enzyme can be used to establish complete in vitro pathways for tRNA modification, allowing systematic analysis of each step in queuosine incorporation.

• Heterologous complementation: As demonstrated with DUF208 genes from various organisms including Dehalococcoides ethenogenes 195, complementation assays in model organisms like E. coli can reveal functional conservation and specificity of pathway components .

• Structure-function studies: Site-directed mutagenesis of conserved residues in recombinant TGT can identify critical catalytic and structural elements, informing mechanisms of substrate recognition and catalysis.

• Substrate specificity analysis: Using various tRNA substrates with recombinant TGT can help define the sequence and structural requirements for efficient modification, potentially revealing differences between bacterial species.

• Cross-species compatibility: Testing the ability of Dehalococcoides sp. TGT to modify tRNAs from diverse organisms can provide insights into the evolution of tRNA modification systems.

What is the significance of studying Dehalococcoides sp. TGT in understanding translation regulation?

Studying Dehalococcoides sp. TGT provides unique insights into translation regulation mechanisms for several reasons:

• Microbiome and environmental adaptation: Dehalococcoides species occupy specialized environmental niches with unique metabolic constraints, making their translation regulation systems potentially adapted to these conditions.

• Evolutionary conservation: Comparing TGT function across diverse organisms including Dehalococcoides species helps identify core conserved features of tRNA modification machinery essential for translation.

• Translation efficiency modulation: Queuosine modifications introduced by TGT are known to influence translation elongation rates at cognate codons (UAC and GAC/GAU) and suppress stop codon readthrough , mechanisms that may be particularly important in organisms with specialized metabolic pathways.

• Proteostasis maintenance: Research has shown that Q-glycosylation contributes to proteostasis, with protein aggregates increasing in cells lacking these modifications . Understanding TGT's role in initiating this modification pathway provides insights into how translation quality control influences cellular health.

• Alternative enzyme pathways: The discovery that organisms can use different enzymes (like QueG and DUF208/QueH) for similar modification steps highlights the diversity of solutions that have evolved for maintaining translation fidelity .

How does disruption of TGT activity affect cellular phenotypes in different organisms?

Disruption of TGT activity leads to various phenotypic consequences across different organisms, revealing the physiological importance of queuosine modifications:

What are the common challenges in expressing active Dehalococcoides sp. TGT and how can they be addressed?

Researchers frequently encounter several challenges when expressing active Dehalococcoides sp. TGT:

• Solubility issues: The enzyme may form inclusion bodies in heterologous expression systems.

  • Solution: Express at lower temperatures (16-20°C), use solubility-enhancing fusion tags (SUMO, MBP), or optimize induction conditions with lower IPTG concentrations.

• Improper folding: The enzyme may be expressed but in an inactive conformation.

  • Solution: Co-express with chaperone proteins, include appropriate metal ions in the growth medium, and ensure reducing conditions during purification.

• Cofactor incorporation: Incomplete incorporation of necessary metal cofactors.

  • Solution: Supplement expression media with relevant metals and consider in vitro reconstitution with Fe/S cluster machinery if applicable.

• Oxygen sensitivity: Potential oxidation of critical cysteine residues.

  • Solution: Perform expression and purification under anaerobic or low-oxygen conditions, maintain reducing environment with DTT or β-mercaptoethanol.

• Protein stability: Rapid degradation or activity loss during storage.

  • Solution: Add stabilizing agents (glycerol, specific buffers), store at -80°C in small aliquots, and avoid freeze-thaw cycles.

How can I differentiate between enzymatic activities of TGT and other enzymes in the queuosine pathway?

Differentiating between the activities of TGT and other enzymes in the queuosine pathway requires specific experimental approaches:

• Substrate specificity: TGT specifically exchanges guanine with preQ₁ at position 34 of tRNAs, while other enzymes like QueA and QueG/QueH act on different substrates (preQ₁-tRNA and oQ-tRNA, respectively) .

• Product analysis: Use analytical techniques such as HPLC, mass spectrometry, or specific chemical labeling to distinguish between different modified nucleosides:

  • TGT activity produces preQ₁-modified tRNA

  • QueA activity converts preQ₁-tRNA to oQ-tRNA

  • QueG/QueH activity converts oQ-tRNA to Q-tRNA

• Genetic complementation: In systems like the E. coli ΔqueG strain, complementation with either queG or duf208 (queH) genes can restore the Q⁺ phenotype, while TGT would not complement this specific defect .

• In vitro reconstitution: Sequential addition of purified enzymes to unmodified tRNA can reveal the specific contribution of each enzyme in the pathway:

  • Start with unmodified tRNA + TGT + preQ₁ → preQ₁-tRNA

  • Then add QueA + SAM → oQ-tRNA

  • Finally add QueG/QueH + cofactors → Q-tRNA

• Cofactor requirements: Different enzymes in the pathway have distinct cofactor requirements that can be used for differentiation:

  • TGT requires Mg²⁺

  • QueA requires S-adenosylmethionine (SAM)

  • QueG requires cobalamin (vitamin B₁₂)

  • QueH may require Fe/S clusters

What advanced techniques are available for studying structure-function relationships in Dehalococcoides sp. TGT?

Several sophisticated techniques can provide deeper insights into structure-function relationships in Dehalococcoides sp. TGT:

• X-ray crystallography and cryo-electron microscopy: These techniques can reveal the three-dimensional structure of TGT alone or in complex with substrate tRNAs, providing atomic-level insights into catalytic mechanisms and substrate recognition.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can identify regions of conformational flexibility and substrate-induced structural changes without requiring protein crystallization.

• Single-molecule FRET: By labeling TGT and its tRNA substrates with appropriate fluorophores, researchers can study the dynamics of enzyme-substrate interactions in real-time.

• Molecular dynamics simulations: Computational approaches can model how Dehalococcoides sp. TGT interacts with substrates and predict the effects of specific mutations.

• Deep mutational scanning: Systematic mutation of TGT residues coupled with functional selection can comprehensively map the sequence-function relationship.

• Cross-linking coupled with mass spectrometry: This approach can identify specific contact points between TGT and its tRNA substrates or between subunits in oligomeric arrangements.

• Ribosome profiling: This technique can assess the global impact of TGT activity on translation dynamics across the transcriptome, as has been done with other components of the queuosine modification pathway .

How do recent findings about queuosine modification impact our understanding of Dehalococcoides sp. TGT function?

Recent discoveries have significantly expanded our understanding of queuosine modification systems and, by extension, the role of TGT enzymes like that from Dehalococcoides species:

• Alternative enzyme pathways: The identification of DUF208 (QueH) as a non-orthologous replacement for QueG in the conversion of epoxyqueuosine to queuosine highlights the evolutionary diversity in this pathway . This suggests that TGT has evolved to function within different downstream processing contexts.

• Functional conservation: Complementation studies showing that duf208 genes from diverse bacteria including Dehalococcoides ethenogenes 195 can functionally replace queG in E. coli demonstrate the modular nature of these pathways .

• Translation regulation: Recent research has revealed that Q-glycosylation slows down elongation at cognate codons and suppresses stop codon readthrough, providing mechanistic insights into how TGT-initiated modifications influence translation dynamics .

• Structural insights: Cryo-EM studies of human ribosome-tRNA complexes have revealed the molecular basis of codon recognition regulated by Q-glycosylations, informing our understanding of how these modifications function at the molecular level .

• Developmental implications: Findings that Q-glycosylation is required for post-embryonic growth in vertebrates highlight the broader physiological importance of the modification pathway initiated by TGT .

• Proteostasis connections: The observation that protein aggregates increase in cells lacking Q-glycosylation establishes connections between tRNA modification and protein quality control systems , suggesting that TGT function has broader implications for cellular health beyond just translation fidelity.

What are the most promising future research directions for Dehalococcoides sp. TGT?

Several promising research directions could significantly advance our understanding of Dehalococcoides sp. TGT:

• Structural characterization: Determining the crystal or cryo-EM structure of Dehalococcoides sp. TGT would provide valuable insights into its specific structural features compared to other bacterial TGTs.

• Substrate engineering: Creating modified tRNAs or nucleobase analogs as substrates for TGT could expand its biotechnological applications or provide tools for studying translation in vivo.

• Environmental adaptation: Investigating how TGT function in Dehalococcoides species relates to their unique environmental niche and metabolic capabilities could reveal connections between translation modification and ecological adaptation.

• Synthetic biology applications: Exploring the use of Dehalococcoides sp. TGT in synthetic biology approaches for controlling translation rates or incorporating modified bases into RNAs for novel functions.

• Systems biology integration: Integrating TGT function into broader models of cellular physiology in Dehalococcoides species to understand how translation regulation interfaces with their distinctive metabolic pathways.

• Comparative enzymology: Systematic comparison of catalytic parameters across TGTs from diverse bacteria could reveal evolutionary adaptations and specialized functions.

How can advanced computational approaches enhance our understanding of Dehalococcoides sp. TGT?

Advanced computational approaches offer powerful tools for studying Dehalococcoides sp. TGT: