Recombinant Clostridium phytofermentans Queuine tRNA-ribosyltransferase (tgt)

What is Queuine tRNA-ribosyltransferase (tgt) and what reaction does it catalyze?

Queuine tRNA-ribosyltransferase (EC 2.4.2.29), also known as tRNA-guanine transglycosylase or guanine insertion enzyme, is an enzyme that catalyzes the base-exchange reaction:

$\text{[tRNA]-guanine + queuine} \rightarrow \text{[tRNA]-queuine + guanine}$

This enzyme belongs to the glycosyltransferase family, specifically the pentosyltransferases. It catalyzes the replacement of the nuclear-encoded guanine base at position 34 (the wobble position) of the anticodon loop in specific tRNAs with the queuine micronutrient .

What are the structural characteristics of Clostridium phytofermentans tgt?

Clostridium phytofermentans tgt is a protein of approximately 378 amino acids. While the specific crystal structure of C. phytofermentans tgt has not been fully characterized in the provided search results, structural studies of related bacterial tgt enzymes (such as from Thermotoga maritima) show that they typically contain zinc binding sites important for catalytic activity. The crystal structure of Thermotoga maritima tgt has been resolved at 1.90 Å resolution, showing a catalytic domain with an active site containing key residues for substrate binding and catalysis .

Which tRNA species are substrates for tgt enzymes?

The human QTRT enzyme displays strict specificity for tRNA species belonging to the G34U35N36 family, which are responsible for decoding the dual synonymous NAU and NAC codons. These include:

• tRNA-Asp

• tRNA-Asn

• tRNA-His

• tRNA-Tyr

The enzyme specifically recognizes tRNAs with GUN anticodons at position 34 (the wobble position) of the anticodon loop. In eukaryotes, both mitochondrial and cytoplasmic tRNAs of these types can be substrates .

How is recombinant C. phytofermentans tgt produced for research purposes?

Recombinant C. phytofermentans tgt can be produced using several expression systems:

• Expression Hosts:

  • E. coli

  • Yeast

  • Baculovirus-infected insect cells

  • Mammalian cell systems

• Purification Approach:

  • Standard recombinant protein methods involving affinity chromatography

  • For long-term storage, the enzyme should be stored at -20°C or -80°C

  • Working aliquots can be kept at 4°C for up to one week

  • Repeated freezing and thawing should be avoided

• Quality Control:

  • Sterile filtration can be performed upon request

  • Low endotoxin preparations are available for sensitive applications

  • Small volumes should be briefly centrifuged to dislodge any liquid in the container's cap before use

What methods are used to assess tgt enzyme activity?

Several experimental approaches are used to study tgt enzyme activity:

• Radioactive Assays: Using tritiated guanine ([³H]guanine) to pre-label tRNA substrates, then measuring displacement by non-radioactive substrates. For example:

  • Pre-labeled tRNATyr (10 μM) is incubated with the QTRT enzyme (100 nM)

  • Test compounds (typically at 50 μM) are assessed for their ability to displace [³H]guanine

  • Data is analyzed as the percentage of [³H]guanine displaced

• Molecular Biology Techniques:

  • Quantitative real-time PCR to assess downstream gene expression effects

  • RNA sequencing to identify modified tRNAs and changes in gene expression profiles

• In vitro Base Exchange Assays:

  • Incubating purified tgt with tRNA substrates and potential queuine analogues

  • Analysis of reaction products by chromatography or mass spectrometry

What is the catalytic mechanism of tgt enzymes?

The tgt enzyme operates through a double-displacement mechanism:

• Nucleophilic Attack: The nucleophile active site attacks the C1' of nucleotide 34 to detach the guanine base from the RNA, forming a covalent enzyme-RNA intermediate involving aspartate 279 (in human QTRT1).

• Substrate Binding: The enzyme first binds queuine, followed by tRNA.

• Base Exchange: The proton acceptor active site deprotonates the incoming queuine (or queuine analog), allowing a nucleophilic attack on the C1' of the ribose.

• Product Formation: This results in the release of Q-modified tRNA (Q-tRNA) and free guanine base.

• Post-modification Processing: After incorporation of preQ1 (7-aminomethyl-7-deazaguanine, a queuine precursor) in bacteria, two additional enzymatic reactions convert it to the final queuosine (Q) modification .

How does substrate specificity differ between bacterial and human tgt enzymes?

Differences in substrate specificity:

The human QTRT enzyme shows remarkable promiscuity in accepting various 7-deazaguanine compounds with different substitutions at the 7-position, while maintaining high specificity for tRNA recognition .

How can tgt be used for incorporating artificial nucleobase analogs into tRNA?

The unique nature of the QTRT enzymatic pathway allows researchers to install artificial nucleobases into the anticodon loop of tRNA:

• Design Strategy: Create 7-deazaguanine derivatives with various substitutions at the 7-position.

• Incorporation Protocol:

  • Incubate purified tgt with target tRNA and the artificial analog

  • The enzyme catalyzes the replacement of guanine with the analog at position 34

  • Multiple analogs can be tested in parallel to assess incorporation efficiency

• Verification Methods:

  • Mass spectrometry to confirm modification

  • Functional assays to assess the impact on tRNA function

• Applications: This approach has been used to create novel tRNA modifications that influence protein translation and have potential therapeutic effects. For example, a 7-deazaguanine derivative (NPPDAG) has been shown to promote recovery in an animal model of multiple sclerosis .

What are the implications of tgt activity for disease research?

Recent research has revealed several disease-related applications of tgt research:

• Autoimmune Disorders: tgt-mediated incorporation of artificial queuine analogs has shown therapeutic potential in multiple sclerosis by:

  • Limiting T-cell proliferation in vitro

  • Curtailing T-helper (Th1 and Th17) responses

  • Modulating cytokine production in both peripheral tissues and central nervous system

  • Re-establishing expression of genes associated with neural repair and regeneration

• Metabolic Disorders: Studies with knockout mice have shown that inhibition of QTRT1 in hepatocytes:

  • Ameliorates hepatic lipogenesis

  • Attenuates hyperlipidemia

  • Reduces liver steatosis

  • Decreases atherosclerotic burden

  • Increases plaque stability in the aorta

  These effects occur primarily through downregulation of de novo lipogenesis without affecting lipoprotein transportation or fatty acid oxidation .

What are the major technical challenges in studying tgt function?

Researchers face several challenges when studying tgt enzymes:

• Protein Expression and Purification:

  • Maintaining enzyme stability during purification

  • Ensuring proper folding in heterologous expression systems

  • Achieving sufficient yields for structural and biochemical studies

• Assay Development:

  • Designing sensitive and specific assays to measure tgt activity

  • Distinguishing between different tRNA substrates

  • Quantifying low-abundance modified tRNAs in complex biological samples

• Structural Studies:

  • Obtaining high-resolution structures of enzyme-substrate complexes

  • Understanding the conformational changes during catalysis

  • Capturing transient intermediates in the reaction pathway

• In vivo Studies:

  • Separating tgt effects from other tRNA modification pathways

  • Controlling for microbiome contributions to queuine availability

  • Establishing appropriate animal models for disease studies

How can researchers identify novel tgt substrates beyond canonical tRNAs?

Methodological approaches for identifying non-canonical substrates:

• Enzyme-RNA Capture-Release Method:

  • Establish a system to capture RNA molecules that interact with tgt

  • Use affinity-tagged enzyme to pull down bound RNAs

  • Sequence captured RNAs to identify potential new substrates

• Structural Analysis:

  • Study minimal RNA elements required for tgt recognition

  • Test chimeric RNA constructs containing recognition elements

• In vivo Crosslinking:

  • Use crosslinking approaches to identify RNA-protein interactions in living cells

  • Compare crosslinked species between wild-type and tgt-deficient cells

• Computational Prediction:

  • Develop algorithms to predict RNA structures that might serve as tgt substrates

  • Validate predictions experimentally through in vitro assays

What is the relationship between tRNA modification by tgt and translation regulation?

The position 34 modification by tgt plays crucial roles in translation:

• Codon Recognition:

  • Queuosine modification at the wobble position affects codon-anticodon interactions

  • This can influence translation rate and accuracy for specific codons

• Translation Dynamics:

  • Modification status may affect ribosome binding and translocation

  • Changes in tRNA modification patterns can alter the speed of translation elongation

• Proteome Effects:

  • Disruption of tgt activity can lead to changes in protein expression profiles

  • These effects may be codon-specific, affecting proteins enriched in certain amino acids

• Experimental Approaches:

  • Ribosome profiling to assess translation efficiency

  • Mass spectrometry to analyze protein abundance changes

  • Codon-specific reporter assays to measure the impact on translation

How conserved is the tgt enzyme across different bacterial species?

The tgt enzyme shows significant conservation across bacterial species:

What structural features determine substrate specificity in tgt enzymes?

Key structural determinants of specificity include:

• Base Recognition Pocket:

  • Human QTRT shows broad ability to recognize various 7-deazaguanine derivatives

  • The binding pocket accommodates different substitutions at the 7-position

• tRNA Recognition Elements:

  • Specificity for G34U35N36 family tRNAs in humans

  • Recognition of the U33G34U35 sequence positioned within a 7-base anticodon loop

  • Requirements for an intact tRNA structure

• Protein Subunits:

  • In eukaryotes, the QTRT enzyme is a complex of two related proteins

  • Catalytic QTRT1 subunit containing the active site

  • Non-catalytic QTRT2 partner that may influence substrate recognition

• Species Differences:

  • Bacterial enzymes can sometimes accept non-physiological substrates

  • Eukaryotic enzymes show higher specificity for canonical tRNA substrates

By understanding these structural determinants, researchers can design targeted experiments to study tgt function and develop potential therapeutic approaches based on this unique enzymatic pathway.

What are the best experimental systems for studying tgt function in vitro?

Optimal experimental systems include:

• Purified Enzyme Systems:

  • Recombinant tgt from E. coli, yeast, baculovirus, or mammalian expression systems

  • In vitro transcribed or purified native tRNA substrates

  • Defined buffer conditions optimized for enzyme activity

• Cell-Free Translation Systems:

  • Rabbit reticulocyte lysate or wheat germ extract supplemented with tgt

  • Allows study of translation effects in a controlled environment

• Fluorescence-Based Assays:

  • Development of fluorescent queuine analogs to monitor incorporation

  • FRET-based systems to track enzyme-substrate interactions

• High-Throughput Screening Platforms:

  • Microplate-based assays for testing multiple substrates or inhibitors

  • Automated systems for large-scale analysis of tgt activity

How can researchers study the impact of tgt activity on cellular function?

Comprehensive approaches include:

• Genetic Manipulation:

  • CRISPR/Cas9-mediated knockout or knockdown of QTRT genes

  • Conditional knockout systems using Cre-lox (as demonstrated in the Qtrt1fl/flAlb-iCre+/- mice)

  • Overexpression systems to study gain-of-function effects

• Transcriptomic Analysis:

  • RNA sequencing to identify changes in gene expression profiles

  • Comparison between wild-type and tgt-deficient cells or tissues

• Proteomic Approaches:

  • Mass spectrometry-based quantitative proteomics

  • Ribosome profiling to assess translation efficiency

  • Pulse-chase experiments to measure protein synthesis rates

• Metabolic Studies:

  • Analysis of lipid metabolism in tgt-deficient systems

  • Measurement of de novo lipogenesis, lipoprotein transportation, and fatty acid oxidation

• Disease Models:

  • Use of appropriate animal models (e.g., hyperlipidemia, atherosclerosis)

  • Administration of artificial queuine analogs to assess therapeutic potential

By employing these methodological approaches, researchers can gain comprehensive insights into the complex roles of Queuine tRNA-ribosyltransferase in cellular function and disease processes.

What are the most promising applications of tgt in therapeutic research?

Based on current findings, the most promising therapeutic applications include:

• Autoimmune Disease Treatment:

  • Development of queuine analogs that can modulate immune responses

  • Targeting specific tRNA modifications to influence T-cell proliferation and cytokine production

  • Potential applications in multiple sclerosis and other autoimmune conditions

• Metabolic Disease Intervention:

  • Targeting QTRT1 to reduce hepatic lipogenesis

  • Development of inhibitors to ameliorate hyperlipidemia and atherosclerosis

  • Potential for treating non-alcoholic fatty liver disease

• Translation Modulation Therapy:

  • Using tgt-mediated tRNA modification to selectively influence translation of specific mRNAs

  • Development of precision medicine approaches based on codon usage patterns

  • Potential applications in genetic diseases with codon-specific effects

What emerging technologies might advance tgt research?

Cutting-edge technologies with potential to transform the field:

• Single-Molecule Techniques:

  • Real-time monitoring of tgt-tRNA interactions

  • Direct observation of base-exchange reactions

  • Understanding enzyme kinetics at unprecedented resolution

• Cryo-EM and Advanced Structural Methods:

  • High-resolution structures of tgt-tRNA-substrate complexes

  • Visualization of conformational changes during catalysis

  • Rational design of substrate analogs and inhibitors

• Nanopore Technology:

  • Direct detection of modified nucleosides in tRNA

  • Real-time monitoring of modification status

  • Potential for diagnostics based on tRNA modification profiles

• Machine Learning Approaches:

  • Prediction of substrate specificity

  • Design of optimal queuine analogs for specific applications

  • Integration of multi-omics data to understand tgt function in complex systems