Recombinant Francisella tularensis subsp. tularensis Queuine tRNA-ribosyltransferase (tgt)
Description
Enzyme Overview
Queuine tRNA-ribosyltransferase (tgt), encoded by the tgt gene, belongs to the EC 2.4.2.29 enzyme class. It catalyzes the insertion of queuine—a hypermodified guanine derivative—into the wobble position of tRNAs for aspartic acid, asparagine, histidine, and tyrosine. This modification is conserved across bacteria, plants, and animals and is linked to stress adaptation and pathogenicity in some microbes .
Recombinant Production
Recombinant tgt from F. tularensis subsp. tularensis is produced in Escherichia coli expression systems. Two primary variants are documented:
Both variants share 100% sequence identity, underscoring conservation across F. tularensis strains.
Critical Research Gaps:
-
No kinetic data (e.g., K~m~, k~cat~) or structural studies (e.g., X-ray crystallography) are available for F. tularensis tgt.
-
Its contribution to tularemia progression remains uncharacterized.
Applications and Research Utility
Recombinant tgt serves as a tool for:
-
Enzymatic Assays: Screening inhibitors targeting queuine modification pathways.
-
Structural Biology: Resolving mechanistic details of tRNA modification.
-
Comparative Studies: Analyzing evolutionary conservation across Francisella subspecies.
Future Directions
Proposed studies include:
-
Knockout Mutant Analysis: Assess tgt’s role in F. tularensis stress response and virulence.
-
Inhibitor Development: Exploit tgt as a therapeutic target given its absence in humans.
-
Structural Characterization: Resolve 3D architecture to guide rational drug design.
Product Specs
Target Background
KEGG: ftw:FTW_1153
Q&A
What is Queuine tRNA-ribosyltransferase (tgt) and what reaction does it catalyze in Francisella tularensis?
Queuine tRNA-ribosyltransferase (EC 2.4.2.29) is an enzyme that catalyzes the exchange of guanine with queuine in specific tRNAs. The specific chemical reaction is:
[tRNA]-guanine + queuine → [tRNA]-queuine + guanine
In this reaction, the two substrates are tRNA-guanine and queuine, while the two products are [tRNA]-queuine and guanine. The enzyme belongs to the family of glycosyltransferases, specifically the pentosyltransferases . The systematic name of this enzyme class is [tRNA]-guanine:queuine tRNA-D-ribosyltransferase. In F. tularensis, this enzyme likely plays a role in translational fidelity and potentially in pathogenicity, though specific studies on its function in this organism remain limited.
How does the structural characterization of tgt inform our understanding of its function?
Extensive structural studies have been conducted on this class of enzymes, with at least 36 structures solved as of late 2007. These include PDB accession codes 1EFZ, 1ENU, 1F3E, 1IQ8, 1IT7, 1IT8, 1J2B, and many others . These structural analyses reveal the binding domains for tRNA and queuine, providing insights into the catalytic mechanism of the enzyme. The structural data enables researchers to understand substrate specificity and design experiments for functional studies of F. tularensis tgt.
What are the key differences between F. tularensis tgt and similar enzymes in other bacterial species?
While specific differences between F. tularensis tgt and other bacterial tgt enzymes are not fully characterized in the available literature, the enzyme belongs to a class of proteins that may show species-specific adaptations. In some bacterial systems, similar enzymes have been characterized as DpdA-like tRNA-guanine transglycosylases . Understanding these differences is crucial for developing targeted approaches in F. tularensis research, particularly when considering the high virulence of F. tularensis subsp. tularensis compared to other subspecies.
What are the optimal conditions for expressing and purifying recombinant F. tularensis tgt?
When expressing recombinant F. tularensis tgt, researchers should consider the following methodological approach:
| Parameter | Recommended Conditions | Considerations |
|---|---|---|
| Expression System | E. coli BL21(DE3) or similar strains | Lower temperature (16-18°C) induction may improve solubility |
| Induction | 0.1-0.5 mM IPTG | Optimize concentration to prevent inclusion body formation |
| Buffer Composition | 50 mM Tris-HCl pH 7.5-8.0, 300 mM NaCl, 5-10% glycerol | Include reducing agents (1-5 mM DTT or β-mercaptoethanol) |
| Purification Method | Ni-NTA affinity chromatography followed by size exclusion | Consider ion exchange chromatography as an additional step |
| Storage | -80°C in buffer with 20-50% glycerol | Aliquot to avoid freeze-thaw cycles |
These conditions should be optimized based on the specific construct design and experimental requirements. Working with F. tularensis proteins requires appropriate biosafety measures due to the pathogenic nature of the organism.
How can enzymatic activity of recombinant F. tularensis tgt be accurately measured?
The enzymatic activity of recombinant F. tularensis tgt can be measured using several complementary approaches:
-
Radioisotope-based assays: Using 3H-labeled guanine or queuine to monitor substrate incorporation into tRNA.
-
HPLC-based methods: Separating modified and unmodified tRNAs to quantify the conversion rate.
-
Mass spectrometry: Detecting mass shifts in tRNA molecules after modification.
-
Fluorescence-based assays: Using fluorescently labeled substrates to monitor real-time kinetics.
Each method provides different advantages in terms of sensitivity, throughput, and information content. For accurate kinetic measurements, researchers should establish standard curves and include appropriate controls to account for non-enzymatic reactions and substrate degradation.
What strategies can be employed for site-directed mutagenesis to study structure-function relationships in F. tularensis tgt?
When designing site-directed mutagenesis experiments for F. tularensis tgt, researchers should:
-
First identify conserved residues through alignment with structurally characterized tgt enzymes
-
Target residues in the putative active site based on structural data from homologous enzymes
-
Consider both conservative and non-conservative substitutions to probe specific chemical properties
-
Use alanine-scanning mutagenesis to identify residues critical for substrate binding versus catalysis
-
Incorporate mutations that mimic phosphorylation (e.g., E/D for S/T) to investigate potential regulatory mechanisms
Following mutagenesis, comprehensive characterization should include protein stability assessments, kinetic analyses, and potentially crystallographic studies of mutant proteins to establish structure-function relationships.
How might tgt contribute to F. tularensis virulence and pathogenicity?
Although direct evidence linking tgt to F. tularensis virulence is limited in the available literature, several hypotheses can be proposed based on the roles of tRNA modification in bacterial pathogenesis:
-
Translational regulation: tgt-mediated tRNA modification may regulate the expression of virulence factors during infection, similar to how F. novicida uses CRISPR/Cas systems to regulate endogenous transcripts encoding immunostimulatory bacterial lipoprotein (BLP) .
-
Stress adaptation: Modified tRNAs could enhance bacterial survival under host-imposed stresses, potentially contributing to the ability of F. tularensis to evade host immune responses.
-
Host-pathogen interactions: tRNA modifications might influence interactions with host cell machinery, potentially affecting intracellular replication capacity.
-
Metabolic adaptation: tgt activity could modulate translational efficiency of specific proteins needed during different stages of infection.
Research examining differential expression or activity of tgt during infection could help elucidate its potential role in virulence, particularly in the context of the highly virulent F. tularensis subsp. tularensis.
How does tgt activity compare between different F. tularensis subspecies and what implications might this have for virulence?
F. tularensis has several subspecies with varying degrees of virulence, including subsp. tularensis (Type A), subsp. holarctica (Type B), subsp. mediasiatica, and subsp. novicida . Comparing tgt activity across these subspecies could provide insights into its potential role in virulence:
| Subspecies | Virulence | Potential tgt Activity Pattern |
|---|---|---|
| tularensis (Type A) | Highest virulence | Potentially optimized activity or regulation |
| holarctica (Type B) | Moderate virulence | Possibly intermediate activity |
| mediasiatica | Lower virulence | May have altered activity or regulation |
| novicida | Least virulent | Could have different substrate specificity |
Experimental approaches to investigate these differences should include:
-
Comparative biochemical characterization of recombinant tgt from each subspecies
-
Analysis of tgt expression levels during infection
-
Generation of tgt mutants in different subspecies to assess virulence impacts
-
Structural studies to identify subspecies-specific features that might correlate with virulence
What potential exists for targeting F. tularensis tgt for antimicrobial development?
The essential nature of specific tRNA modifications and the structural differences between bacterial and eukaryotic tgt enzymes make F. tularensis tgt a potential target for selective antimicrobial development. Research approaches could include:
-
High-throughput screening for selective inhibitors of F. tularensis tgt
-
Structure-based drug design leveraging the available crystallographic data for tgt enzymes
-
Evaluation of identified inhibitors against intact F. tularensis under biosafety conditions
-
Assessment of synergistic effects when combined with existing antibiotics
The development of tgt inhibitors could provide novel therapeutic options for tularemia, particularly important given the classification of F. tularensis as a category A agent of bioterrorism .
What are common challenges in obtaining enzymatically active recombinant F. tularensis tgt and how can they be addressed?
Researchers frequently encounter several challenges when working with recombinant F. tularensis tgt:
| Challenge | Potential Solutions |
|---|---|
| Low solubility | - Use solubility-enhancing fusion tags (MBP, SUMO) - Optimize induction conditions (lower temperature, reduced IPTG) - Screen multiple buffer conditions |
| Poor enzymatic activity | - Ensure proper cofactor inclusion - Check for inhibitory compounds in purification buffers - Verify proper folding using circular dichroism |
| Protein instability | - Add stabilizing agents (glycerol, reducing agents) - Identify and mutate surface-exposed cysteines - Optimize pH and ionic strength |
| Substrate availability | - Develop in-house synthesis of queuine - Establish collaborations with chemical biology groups - Consider commercial sources where available |
Systematic optimization of these parameters is essential for obtaining functionally active enzyme preparations suitable for biochemical and structural studies.
What biosafety considerations are critical when working with recombinant proteins from F. tularensis?
F. tularensis is classified as a Tier 1 Select Agent due to its high pathogenicity and potential for bioterrorism applications . While recombinant proteins themselves may not pose the same risk as the intact organism, researchers should adhere to strict biosafety guidelines:
-
Risk assessment: Conduct thorough risk assessments before beginning work with F. tularensis-derived proteins
-
Containment measures: Consider appropriate biosafety level requirements based on institutional guidelines
-
Decontamination protocols: Establish effective decontamination procedures for all equipment and materials
-
Personnel training: Ensure all researchers are trained in proper handling techniques
-
Documentation: Maintain detailed records of all work with F. tularensis-derived materials
Additionally, researchers should verify that recombinant protein preparations are free from contaminating F. tularensis cells or endotoxins that could pose safety risks or confound experimental results.
How might new technologies advance our understanding of F. tularensis tgt function in pathogenesis?
Emerging technologies that could advance understanding of F. tularensis tgt include:
-
CRISPR-Cas9 genome editing: For precise manipulation of the tgt gene in F. tularensis to assess function
-
Ribosome profiling: To examine translational effects of tgt-mediated tRNA modifications
-
Single-cell RNA-seq: To investigate heterogeneity in tgt expression during infection
-
Cryo-EM: For high-resolution structural studies of tgt-tRNA complexes
-
Metabolomics: To assess the impact of tgt activity on the bacterial metabolome
Integration of these approaches could provide a systems-level understanding of how tgt contributes to F. tularensis biology and pathogenicity, potentially identifying new avenues for therapeutic intervention.
What is the relationship between tgt and other virulence mechanisms in F. tularensis?
Future research should investigate potential interactions between tgt and other known virulence factors in F. tularensis, such as Type IV pili that contribute to host cell adhesion and virulence . The relationship between tRNA modification and other regulatory systems, such as the CRISPR/Cas system that has been implicated in virulence in F. novicida , could reveal integrated virulence networks. These studies would help place tgt function in the broader context of F. tularensis pathogenicity and potentially identify synergistic targets for therapeutic development.
