Recombinant Nitratiruptor sp. Queuine tRNA-ribosyltransferase (tgt)

What is Nitratiruptor sp. Queuine tRNA-ribosyltransferase and what ecological niche does it occupy?

Nitratiruptor sp. Queuine tRNA-ribosyltransferase (tgt) is an enzyme (EC 2.4.2.29) isolated from Nitratiruptor sp. SB155-2, an ε-Proteobacteria found in deep-sea hydrothermal vent environments. This extremophile was isolated from hydrothermal vents at approximately 1,000 meters depth in the Hatoma Knoll hydrothermal field in the southern-Okinawa Trough, Japan . As a member of the Nitratiruptor genus, this organism represents one of the most numerically abundant chemolithoautotrophic Campylobacterota populations in the mixing zones between hydrothermal fluids and ambient seawater .

The enzyme catalyzes the base-exchange reaction where guanine is replaced by queuine or its precursors in the anticodon loop of specific tRNAs. This adaptation to extreme conditions likely contributes to the unique properties of Nitratiruptor sp. tgt compared to homologous enzymes from mesophilic organisms .

What is the biochemical function of tgt enzyme and what substrates does it recognize?

The tgt enzyme catalyzes an irreversible base-for-base exchange reaction, replacing guanine with queuine in tRNA molecules . This reaction occurs specifically at position 34 (the wobble position) of tRNAs with G34U35N36 anticodons, which correspond to tRNAs for aspartic acid (Asp), asparagine (Asn), tyrosine (Tyr), and histidine (His) .

The enzyme follows a ping-pong mechanism involving:

• Formation of a covalent enzyme-RNA intermediate

• Release of the displaced guanine

• Nucleophilic attack by queuine to form the modified tRNA

• Release of the Q-modified tRNA

While bacterial TGTs primarily incorporate preQ1 (a queuine precursor), which requires further enzymatic steps to form Q-modified tRNA, eukaryotic TGTs directly incorporate queuine to form Q-tRNA .

How do bacterial and eukaryotic tgt enzymes differ structurally and functionally?

Bacterial and eukaryotic tgt enzymes exhibit significant structural and functional differences as summarized in this comparative table:

These differences reflect the divergent evolution of these enzymes and their adaptation to different physiological contexts .

What expression systems and purification methods are optimal for recombinant Nitratiruptor sp. tgt?

For efficient expression and purification of recombinant Nitratiruptor sp. tgt, the following protocol has been shown to be effective :

Expression System:

• Host strain: E. coli BL21(DE3)

• Expression vector: pCold I or similar low-temperature induction vectors

• Induction conditions: Optimization required, but cold-shock induction (15-16°C) is often beneficial for extremophile proteins

Purification Strategy:

• Affinity chromatography using His-tag (N-terminal or C-terminal)

• Size exclusion chromatography to ensure homogeneity

• For higher purity (>95%), ion exchange chromatography can be employed as a polishing step

Storage Conditions:

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (50% is recommended default)

• Store at -20°C for short term, -80°C for long-term storage

• Avoid repeated freeze-thaw cycles

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

What assays can be used to measure Nitratiruptor sp. tgt activity?

Several complementary assays can be employed to measure tgt activity, each providing different insights into enzyme function:

1. tRNA [14C] Guanine Displacement Assay:
This assay monitors the displacement of radiolabeled guanine from pre-charged tRNA:

• Pre-label tRNA with [14C] guanine using E. coli TGT

• Incubate labeled tRNA with tgt enzyme and test compounds

• Separate tRNA from free nucleobases using DEAE cellulose

• Quantify displaced [14C] guanine by scintillation counting

2. tRNA [14C] Guanine Incorporation Assay:
This assay measures the reversibility of tgt-catalyzed modifications:

• Modify tRNA with non-labeled nucleobases using tgt

• Incubate with [14C] guanine and tgt

• Measure incorporation of [14C] guanine into tRNA

3. Cellular Incorporation Assays:
For measuring activity in cellular contexts:

• Culture cells with various concentrations of queuine or analogs

• Add [3H] queuine as a tracer

• Harvest cells, prepare TCA precipitates

• Quantify incorporation by scintillation counting

4. Northern Blot with APB Gels:
For detecting Q-modified tRNAs:

• Separate tRNAs on polyacrylamide gels containing 3-(acrylamido)phenylboronic acid (APB)

• Q-modified tRNAs migrate more slowly

• Transfer to nylon membrane and detect with biotinylated probes specific for tRNAs of interest

What is the substrate specificity of Nitratiruptor sp. tgt and how does it compare to other TGTs?

Nitratiruptor sp. tgt, like other bacterial TGTs, likely recognizes specific tRNAs with G34U35N36 anticodons and can accommodate various 7-deazaguanine derivatives as substrates. While specific data for Nitratiruptor sp. tgt is limited, studies with related TGTs provide insights into substrate preferences:

tRNA Substrates:

• tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis with G34U35N36 anticodons

• The minimal recognition element appears to be a structured hairpin containing the target G nucleobase in a "UGU" loop motif

Nucleobase Substrates:
Compound activity relative to the natural substrate can be summarized as:

The irreversibility of queuine and its analogues is attributed to the substitution of C-7 for N-7, which prevents the reverse reaction .

Can tgt enzymes modify DNA substrates and what factors influence this activity?

While tgt enzymes primarily modify RNA, research has demonstrated that they can also modify specific DNA substrates under certain conditions. This was initially thought impossible, but recent studies have shown it is feasible with optimized substrates .

Key factors that enable DNA modification by TGT include:

• Recognition Element Modification: Converting the minimal "TGT" loop motif in DNA to "TGdU" or "dUGdU" significantly enhances modification efficiency

• Structural Constraints: Controlling steric constraints in DNA hairpins dramatically affects labeling efficiency. When optimized, near-quantitative site-specific modification can be achieved

• Hairpin Design: The stem structure supporting the loop is less critical than the loop sequence itself, as studies have shown that various stem sequences can support efficient labeling if paired with the right loop

This DNA-modifying capability has practical applications in rapidly synthesizing probes for fluorescent Northern blotting and RNA FISH visualization .

What is the biological significance of queuosine modification in tRNAs?

Queuosine (Q) modification at position 34 of specific tRNAs plays several important biological roles:

• Translation Regulation: Q modification influences translational efficiency and accuracy, controlling the speed of Q-decoded codons and near-cognate codons

• Cellular Homeostasis: This modification is involved in various cellular processes including:

  • Cell proliferation regulation

  • Stress response mechanisms

  • Cell signaling pathways

• Disease Associations: Deficiencies in Q-tRNA levels correlate with several pathological conditions:

  • Tumor growth and cancer progression

  • Autoimmune diseases like experimental autoimmune encephalomyelitis

  • Leukemia and other hematological disorders

• Metabolic Regulation: Recent research indicates that Q modification enzymes (particularly QTRT1) may be involved in lipid metabolism:

  • Inhibition of QTRT1 in hepatocytes ameliorates hepatic lipogenesis

  • This inhibition reduces atherosclerosis and hyperlipidemia in mouse models

• Microbiome Interactions: Q and its precursors represent micronutrients that may be competed for by hosts and their microbiota, suggesting a role in host-microbe interactions

What therapeutic and biotechnological applications have been developed using tgt enzymes?

TGT enzymes have shown promising applications in both therapeutic and biotechnological contexts:

Therapeutic Applications:

• Autoimmune Disease Treatment: An artificial queuine analogue (NPPDAG) incorporated by tgt showed remarkable efficacy in an animal model of multiple sclerosis:

  • Induced rapid and complete remission of ongoing disease

  • Maintained normal naive T cell populations while targeting pathogenic T cells

  • Effect was TGT-dependent, confirming the mechanism

• Metabolic Disorder Treatment: Inhibition of QTRT1 (the eukaryotic TGT catalytic subunit) has shown promise in treating metabolic disorders:

  • Ameliorated hyperlipidemia and liver steatosis

  • Reduced atherosclerotic burden while increasing plaque stability

  • Significantly downregulated de novo lipogenesis

Biotechnological Applications:

• RNA Labeling (RNA-TAG): TGT enzymes can be used to site-specifically label RNA molecules:

  • Exchange target G for chemically modified preQ1 substrates linked to biotin or fluorophores

  • Enables visualization and isolation of specific RNA species

• DNA Labeling (DNA-TAG): Optimized TGT-based system for labeling DNA:

  • Near-quantitative site-specific modification of DNA substrates

  • Rapid synthesis of probes for fluorescent Northern blotting

  • Development of RNA FISH visualization probes

• Nucleic Acid Modification: The ability to incorporate synthetic nucleobases into specific positions in nucleic acids opens possibilities for:

  • Studying RNA modification effects on translation

  • Developing novel biosensors

  • Creating nucleic acids with non-natural functions

How do environmental factors affect the stability and activity of Nitratiruptor sp. tgt?

As an enzyme from a deep-sea extremophile, Nitratiruptor sp. tgt likely possesses unique adaptations to extreme conditions. While specific data for the tgt enzyme is limited, insights can be drawn from studies of other Nitratiruptor sp. enzymes and the organism's native environment:

Environmental Adaptations:

• Temperature: Nitratiruptor sp. grows optimally at 55°C with a growth range of 40-57°C . Its enzymes, including tgt, likely show thermostability and optimal activity at elevated temperatures.

• pH: The organism grows optimally at pH 6.4 with a range of 5.4-6.9 , suggesting its enzymes function best under slightly acidic conditions.

• Salt Concentration: Optimal growth occurs at 2.5% (w/v) NaCl with a range of 1.5-4.0% . High salt concentrations may be required for optimal enzyme activity.

Comparative Enzyme Data:
NitAly, an alginate lyase from Nitratiruptor sp., demonstrates:

• Optimum temperature of 70°C

• Optimum pH around 6

• Requirement for high NaCl concentration (0.8-1.4 M) for maximum activity

• 50% activity loss after 30 minutes at 67°C

• Heat stability dependent on disulfide bonds (Cys-80 and Cys-232)

These properties likely reflect adaptations to the deep-sea hydrothermal vent environment and may be shared by Nitratiruptor sp. tgt, informing optimal experimental conditions for researchers.

What insights can be gained from studying Nitratiruptor sp. tgt for engineering tgt enzymes with novel properties?

Studying Nitratiruptor sp. tgt offers several valuable insights for enzyme engineering:

• Thermostability Mechanisms: Understanding how this enzyme maintains stability at high temperatures can inform strategies to enhance the stability of mesophilic tgt enzymes:

  • Potential disulfide bonds (as seen in NitAly)

  • Higher proportion of hydrophobic core residues

  • Surface charge distribution adaptations

• Substrate Promiscuity: Extremophile enzymes often show broader substrate ranges due to more flexible active sites, potentially informing the design of tgt variants with expanded substrate scopes

• Structure-Function Relationships: Comparing Nitratiruptor sp. tgt with mesophilic homologs can reveal:

  • Critical residues for catalysis versus stability

  • Regions tolerant to mutation versus conserved functional domains

  • Substrate binding pocket adaptations that could be transferred to other tgt enzymes

• Novel Catalytic Properties: The unique environmental adaptations may confer novel catalytic properties that could be harnessed for biotechnological applications:

  • Enhanced reaction rates under extreme conditions

  • Different substrate preferences

  • Altered product distributions

Understanding these features can guide rational design of tgt variants with improved properties for research, biotechnology, and therapeutic applications.

What are common challenges when working with recombinant Nitratiruptor sp. tgt and how can they be addressed?

Researchers working with recombinant Nitratiruptor sp. tgt may encounter several challenges:

1. Expression and Solubility Issues:

• Challenge: Low expression levels or insoluble protein formation

• Solutions:

  • Optimize growth temperature (try lower temperatures like 15-20°C)

  • Use specialized expression strains (e.g., ArcticExpress)

  • Co-express with chaperones

  • Test different fusion tags (MBP, SUMO, GST)

  • Include stabilizing additives (e.g., glycerol, specific ions)

2. Activity and Stability:

• Challenge: Low activity or rapid inactivation under standard conditions

• Solutions:

  • Include higher salt concentrations (0.8-1.4 M NaCl) in reaction buffers

  • Maintain slightly acidic pH (around pH 6.4)

  • Add reducing agents cautiously (may affect disulfide bonds)

  • Test activity at elevated temperatures (40-70°C)

3. Substrate Recognition:

• Challenge: Poor recognition of standard tRNA substrates

• Solutions:

  • Use tRNAs with optimized hairpin structures

  • Test various G34U35N36 anticodon-containing tRNAs

  • Consider modified minimal recognition elements

4. Assay Sensitivity:

• Challenge: Difficulty detecting low-level enzyme activity

• Solutions:

  • Use radiolabeled assays for highest sensitivity ([14C] or [3H])

  • Optimize reaction conditions based on extremophile parameters

  • Consider fluorescent detection methods with low background

How can researchers optimize experimental design when studying the incorporation of artificial nucleobases by Nitratiruptor sp. tgt?

When studying artificial nucleobase incorporation by Nitratiruptor sp. tgt, consider these optimization strategies:

1. Nucleobase Design Principles:

• Maintain the amino nitrogen at position 2 (critical for recognition)

• Consider modifications at the 7-position (C-7 substitution for N-7 prevents reversibility)

• Balance size and chemical properties to maintain enzyme recognition while introducing novel functionality

2. Reaction Optimization:

• Temperature: Test gradient from 37-70°C

• pH: Optimize within pH 5.5-7.0 range

• Salt: Include NaCl at 0.8-1.4 M

• Time: Monitor reaction progress from 30 min to 24 hours

• Enzyme:substrate ratio: Typically 1:10 to 1:100 molar ratio

3. Analysis Methods:

• Incorporation rate: Use radiolabeled nucleobases or tRNAs

• Modified tRNA identification: Northern blotting with APB gels

• Modification site confirmation: Mass spectrometry or sequence-specific cleavage

• Functional impact: Translation assays with modified tRNAs

4. Controls and Validation:

• Include natural substrate controls (preQ1, queuine)

• Use known poor substrates as negative controls

• Confirm site-specificity of incorporation

• Validate using multiple detection methods

By systematically optimizing these parameters, researchers can enhance the efficiency and specificity of artificial nucleobase incorporation by Nitratiruptor sp. tgt.