Recombinant Desulfotomaculum reducens Queuine tRNA-ribosyltransferase (tgt)

Introduction

Recombinant Desulfotomaculum reducens Queuine tRNA-ribosyltransferase (TGT) is an engineered enzyme derived from the Gram-positive, sulfate-reducing bacterium Desulfotomaculum reducens MI-1. This enzyme belongs to the tRNA-guanine transglycosylase family, which catalyzes the exchange of guanine at the wobble position (position 34) of specific tRNAs with queuine, a hypermodified 7-deazaguanine derivative. This modification enhances tRNA stability and translational fidelity, particularly under stress conditions such as nutrient limitation .

Enzyme Composition and Mechanism

• Subunit Architecture: Unlike eukaryotic TGTs, which function as heterodimers (e.g., human QTRT1-QTRTD1 ), bacterial TGTs like D. reducens TGT are typically homodimeric proteins. The catalytic core resembles the (β/α)₈-barrel fold observed in Zymomonas mobilis TGT, with a zinc-binding subdomain critical for tRNA recognition .

• Catalytic Activity: TGT facilitates a base-exchange reaction, replacing guanine-34 with queuine or its precursor preQ₁ in a two-step transglycosylation process. This requires a covalent tRNA-enzyme intermediate .

Recombinant Expression and Purification

Recombinant D. reducens TGT is produced via heterologous expression in Escherichia coli. Key steps include:

1. Gene Cloning: The tgt gene (homolog of queT or qtrt1) is cloned into an expression vector under an inducible promoter.

2. Co-Expression: For functional activity, co-expression with accessory proteins (e.g., sulfur relay enzymes) may be required, as seen in Bacillus subtilis MnmA-YrvO systems .

3. Affinity Purification: Polyhistidine tags enable Ni²⁺-based purification, followed by size-exclusion chromatography to isolate the active dimer .

Substrate Specificity and Kinetics

• Metal Dependence: Activity is inhibited by Mg²⁺ and Mn²⁺ at >1 mM concentrations, contrasting with archaeal homologs .

• RNA Recognition: The anticodon stem-loop (U33G34U35) is sufficient for binding, as demonstrated using minimal RNA substrates .

Role in Bacterial Physiology

• Stress Adaptation: Queuine-modified tRNAs improve translational accuracy during sulfur starvation, a critical survival strategy for D. reducens in metal-rich environments .

• Sulfur Metabolism: TGT activity depends on sulfur mobilizing enzymes like cysteine desulfurases (e.g., YrvO in B. subtilis), which provide the thiol groups for queuine biosynthesis .

Comparative Analysis with Homologs

Applications and Biotechnological Potential

• Biomarker Development: Queuosine-modified tRNA levels correlate with cellular stress responses, offering diagnostic potential .

• Antibiotic Targets: Bacterial TGTs are explored as targets for antimicrobials due to their absence in humans .

Challenges and Future Directions

• Structural Resolution: No crystal structure of D. reducens TGT is available; homology modeling based on Z. mobilis TGT (PDB: 7NQ4 ) is currently used.

• In Vivo Function: The interplay between TGT and sulfur metabolism in D. reducens remains poorly characterized .