Recombinant Sorangium cellulosum Queuine tRNA-ribosyltransferase (tgt)

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

Queuine tRNA-ribosyltransferase (EC 2.4.2.29) is an enzyme belonging to the glycosyltransferase family, specifically the pentosyltransferases. This enzyme catalyzes a critical base-exchange reaction in tRNA processing, where it exchanges a guanine (G) residue at the wobble position (position 34) of the anticodon with queuine in tRNAs with GU(N) anticodons . The chemical reaction can be represented as:

[tRNA]-guanine + queuine → [tRNA]-queuine + guanine

This reaction is fundamental to the formation of queuosine, a hypermodified nucleoside found in tRNAs specific for aspartic acid, asparagine, histidine, and tyrosine . The enzyme's activity is essential for proper tRNA function and, consequently, for the fidelity and efficiency of protein synthesis. In bacterial systems like Sorangium cellulosum, this enzyme contributes to the complex machinery regulating translation through tRNA modification.

How should Recombinant Sorangium cellulosum Queuine tRNA-ribosyltransferase be stored and handled in laboratory settings?

Optimal storage and handling protocols for Recombinant Sorangium cellulosum Queuine tRNA-ribosyltransferase are essential for maintaining enzyme activity and ensuring experimental reproducibility. The following methodological guidelines should be observed:

• Storage temperature: Store the protein at -20°C for regular use, or at -80°C for extended storage to prevent degradation .

• Reconstitution procedure:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration between 5-50% (with 50% being optimal for most applications)

  • Aliquot the reconstituted protein for long-term storage at -20°C/-80°C

• Freeze-thaw considerations: Repeated freezing and thawing should be avoided as it can compromise protein integrity and activity. Working aliquots can be stored at 4°C for up to one week .

• Shelf life expectations: The liquid form typically has a shelf life of approximately 6 months when stored at -20°C/-80°C, while the lyophilized form can remain viable for up to 12 months under the same storage conditions .

These handling procedures are designed to preserve the structural integrity and enzymatic activity of the recombinant protein, thereby ensuring optimal performance in experimental applications.

What are the common nomenclature and classification systems for Queuine tRNA-ribosyltransferase?

Queuine tRNA-ribosyltransferase is identified and classified through several standardized systems in biochemistry and enzymology:

Enzyme Commission (EC) Classification:

• EC Number: 2.4.2.29

• Class 2: Transferases

• Subclass 4: Glycosyltransferases

• Sub-subclass 2: Pentosyltransferases

Alternative Nomenclature:

• tRNA-guanine transglycosylase

• Guanine insertion enzyme

• tRNA transglycosylase

• Q-insertase

• Queuine transfer ribonucleate ribosyltransferase

• Transfer ribonucleate glycosyltransferase

• tRNA guanine transglycosidase

• Guanine, queuine-tRNA transglycosylase

Systematic Name:

• [tRNA]-guanine:queuine tRNA-D-ribosyltransferase

Database Identifiers:

• CAS Registry Number: 72162-89-1

• UniProt ID for Sorangium cellulosum: A9FI06

Understanding this nomenclature is crucial for literature searches, database queries, and comparing findings across different research publications. The variety of names reflects the enzyme's discovery history and functional characterization by different research groups.

What is the catalytic mechanism of Queuine tRNA-ribosyltransferase at the molecular level?

Queuine tRNA-ribosyltransferase operates through a sophisticated double-displacement mechanism that involves multiple steps and active site residues. The detailed catalytic process occurs as follows:

• Initial binding phase: The enzyme recognizes and binds to specific tRNAs with GU(N) anticodons (tRNA-Asp, -Asn, -His, and -Tyr) .

• Nucleophilic attack: A nucleophile in the enzyme's active site attacks the C1' position of nucleotide 34 (the wobble position) in the tRNA anticodon, resulting in the detachment of the guanine base from the RNA backbone .

• Formation of covalent intermediate: This nucleophilic attack leads to the formation of a covalent enzyme-RNA intermediate, which stabilizes the reaction complex .

• Substrate deprotonation: A proton acceptor in the active site deprotonates the incoming queuine (or its precursor PreQ1 in some systems), activating it for the subsequent reaction step .

• Second nucleophilic attack: The deprotonated queuine performs a nucleophilic attack on the C1' of the ribose, displacing the enzyme and forming the product .

• Post-incorporation modifications: In systems where PreQ1 is incorporated instead of queuine, additional enzymatic reactions convert PreQ1 to queuine (Q), ultimately resulting in the formation of the hypermodified nucleoside queuosine .

This elaborate mechanism ensures the precise exchange of nucleobases at a specific position in the tRNA, maintaining the fidelity of this critical modification. The crystal structures available in the PDB provide valuable insights into the spatial arrangement of the active site residues and their interactions with substrates during catalysis .

How does Q-glycosylation affect translational processes and proteostasis?

Q-glycosylation, the addition of sugar moieties to queuosine in tRNAs, has profound effects on translational processes and cellular proteostasis. Recent research has revealed several key mechanisms through which these modifications influence protein synthesis:

• Translational speed regulation: Q-glycosylation slows down the elongation rate at cognate codons. Specifically, galactosylation affects the UAC codon, while mannosylation impacts the GAC (and GAU) codons . This controlled deceleration is thought to facilitate proper protein folding by providing additional time for nascent peptides to adopt correct conformations.

• Suppression of stop codon readthrough: Galactosylation of queuosine has been demonstrated to suppress stop codon readthrough, enhancing the fidelity of translation termination . This function is critical for preventing the production of extended proteins with potentially deleterious effects.

• Proteostasis maintenance: Cells lacking Q-glycosylation show increased accumulation of protein aggregates, indicating that these modifications play an essential role in maintaining proteostasis . The connection between translation rate and protein folding efficiency appears to be a key factor in this relationship.

• Developmental impact: In vertebrate models, specifically zebrafish, knockout of the enzymes responsible for Q-glycosylation (qtgal and qtman) resulted in shortened body length, suggesting that these modifications are required for proper post-embryonic growth . This observation links tRNA modification to broader developmental processes.

The molecular basis for these effects has been elucidated through cryo-electron microscopy of human ribosome-tRNA complexes, which revealed how Q-glycosylations regulate codon recognition at the molecular level . These findings highlight the sophisticated role of tRNA modifications in fine-tuning the translation apparatus and maintaining cellular proteostasis.

What experimental approaches can be used to study the function of Queuine tRNA-ribosyltransferase in vitro?

Investigating the function of Queuine tRNA-ribosyltransferase in vitro requires specialized methodologies that target various aspects of the enzyme's activity and structural properties. The following experimental approaches are particularly valuable:

• Enzymatic activity assays:

  • Base-exchange assays using radiolabeled substrates to track the incorporation of queuine into tRNA

  • Spectrophotometric methods monitoring guanine release

  • HPLC analysis of modified and unmodified tRNAs

• Protein production and purification:

  • Recombinant expression in E. coli systems, as demonstrated with Sorangium cellulosum Queuine tRNA-ribosyltransferase

  • Affinity chromatography using histidine or other tags

  • Size-exclusion chromatography for final purification and buffer exchange

• Structural analysis:

  • X-ray crystallography to determine three-dimensional structure

  • Cryo-electron microscopy to visualize enzyme-tRNA complexes

  • NMR spectroscopy for dynamics studies

• Binding studies:

  • Isothermal titration calorimetry (ITC) to measure binding affinities

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Fluorescence anisotropy to assess protein-RNA interactions

• Reconstitution experiments:

  • In vitro reconstitution of Q-glycosylation using purified enzymes and nucleotide diphosphate sugars

  • Step-wise assembly of the enzyme complex when multiple subunits are involved

For example, researchers have successfully employed biochemical approaches to identify RNA glycosylases (QTGAL and QTMAN) and reconstitute Q-glycosylation of tRNAs using nucleotide diphosphate sugars . These methodologies provide powerful tools for dissecting the enzyme's function, substrate specificity, and catalytic mechanism in controlled laboratory conditions.

What are the comparative differences between Queuine tRNA-ribosyltransferase from Sorangium cellulosum and other species?

Queuine tRNA-ribosyltransferase exhibits notable evolutionary conservation but also displays species-specific differences that reflect adaptations to different cellular environments. A comparative analysis reveals several key distinctions:

A particularly significant difference is that in human cells, queuine tRNA-ribosyltransferase functions as a heterodimer, with QTRT-1 interacting with QTRTD1 to form the active enzyme complex . This contrasts with the single-subunit enzyme found in Sorangium cellulosum . Additionally, while eukaryotic systems generally use queuine directly, bacterial systems typically incorporate preQ1, which is then converted to queuosine through additional enzymatic steps .

These comparative differences provide valuable insights into the evolution of this enzyme family and can inform experimental design when using recombinant enzymes from different species as research tools or therapeutic targets.

How can knockout models be effectively utilized to study Queuine tRNA-ribosyltransferase function in vivo?

Knockout models have emerged as powerful tools for investigating the physiological relevance of Queuine tRNA-ribosyltransferase and related enzymes in vivo. Effective experimental strategies include:

• Cell line knockout approaches:

  • CRISPR-Cas9 genome editing to create precise knockouts of tgt genes

  • Analysis of translational changes through ribosome profiling

  • Assessment of proteostasis through protein aggregation assays

  • Rescue experiments with wild-type or mutant versions of the enzyme

• Animal model development:

  • Generation of zebrafish qtgal and qtman knockout lines to study developmental effects

  • Phenotypic characterization focused on growth parameters and morphology

  • Tissue-specific expression analysis of tgt and related genes

  • Molecular phenotyping through transcriptomics and proteomics

• Phenotypic analysis methodologies:

  • Morphometric measurements (e.g., body length in zebrafish models)

  • Developmental timing assessment

  • Tissue-specific effects through histological examination

  • Behavioral assays for neurological phenotypes

• Molecular consequence analysis:

  • Ribosome profiling to measure translational rates at specific codons

  • Mass spectrometry to identify changes in protein expression and modification

  • RNA sequencing to detect compensatory mechanisms

  • Polysome profiling to assess global translation efficiency

Recent research utilizing knockout models has revealed significant physiological insights, such as the finding that zebrafish lacking Q-glycosylation enzymes display shortened body length, suggesting these modifications are required for proper post-embryonic growth in vertebrates . Additionally, ribosome profiling of knockout cells demonstrated that Q-glycosylation slows down elongation at specific codons, providing a mechanistic explanation for the observed phenotypes .

What are the potential research applications of recombinant Queuine tRNA-ribosyltransferase in RNA modification studies?

Recombinant Queuine tRNA-ribosyltransferase offers diverse applications in RNA modification research, spanning from fundamental mechanistic studies to therapeutic explorations:

• Synthetic biology applications:

  • Engineering tRNAs with site-specific modifications to study translation dynamics

  • Creating artificial translation systems with controlled modification states

  • Developing biosensors for RNA modification states in complex samples

• Structural biology approaches:

  • Co-crystallization with substrate analogs to elucidate binding mechanisms

  • Time-resolved structural studies to capture catalytic intermediates

  • Structure-guided design of inhibitors or activity modulators

• Disease model investigations:

  • Exploring the relationship between tRNA modifications and neurodegenerative diseases

  • Investigating cancer-associated alterations in tRNA modification patterns

  • Studying the role of queuosine in microbial pathogenesis

• Methodological innovations:

  • Development of in vitro reconstitution systems for studying tRNA modification pathways

  • Creation of chemoenzymatic methods for site-specific RNA labeling

  • Implementation of high-throughput screening platforms for modulators of enzyme activity

• Translational regulation studies:

  • Manipulation of translation rates through controlled tRNA modification

  • Investigation of codon-specific effects on protein folding

  • Analysis of stop codon readthrough suppression mechanisms

The successful reconstitution of Q-glycosylation using purified enzymes and nucleotide diphosphate sugars represents a significant methodological advancement in this field . Such systems enable detailed mechanistic studies of the enzymes involved and provide platforms for investigating the functional consequences of these modifications in controlled environments.