Recombinant Nitrosomonas europaea Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the structural composition of Nitrosomonas europaea phenylalanyl-tRNA synthetase?

Nitrosomonas europaea phenylalanyl-tRNA synthetase (PheRS) is a multidomain (alphabeta)2 heterotetrameric protein responsible for synthesizing Phe-tRNA(Phe) during protein synthesis. The alpha subunit (encoded by pheS) forms the catalytic core of the enzyme, while the beta subunit (encoded by pheT) contains several autonomous structural modules with diverse functions. These functions include tRNA anticodon binding and editing of misaminoacylated species Tyr-tRNA(Phe) . The quaternary structure consists of two alpha and two beta subunits arranged in a specific orientation that facilitates both aminoacylation and editing activities.

Where is the pheT gene located in the Nitrosomonas europaea genome?

The pheT gene, encoding the beta subunit of phenylalanyl-tRNA synthetase, is located in the 15.5-kb intergenic spacer region between ammonia monooxygenase (amo) and hydroxylamine oxidoreductase (hao) gene clusters in the Nitrosomonas europaea genome. This region also contains other important genes including threonyl tRNA synthetase (thrS), initiation factor 3 (infC), ribosomal protein L20 (rplT), and the alpha subunit of phenylalanyl-tRNA synthetase (pheS) . This genomic organization is conserved between N. europaea strain ATCC 19718 and Nitrosomonas sp. strain ENI-11, suggesting functional importance in ammonia-oxidizing bacteria.

How does the B2 domain of the pheT subunit contribute to PheRS function?

The B2 domain of the beta subunit (pheT) functions as a secondary tRNA-binding site that contributes to the editing process. Research comparing full-length PheRS with PheRS lacking the B2 domain (PheRS deltaB2) revealed that this domain promotes the translocation of mischarged tRNA to the editing site of PheRS. When the B2 domain is deleted, there is a 2-fold decrease in the catalytic efficiency (kcat/KM) of Tyr-tRNA(Phe) hydrolysis . Structurally, the B2 domain is a homologue of the EMAPII/OB fold, which has been shown in other systems to contribute to tRNA binding. This role is consistent with previous studies suggesting that the highly conserved EMAPII fold modulates the affinity of tRNA for its primary binding site.

What are the optimal conditions for expressing recombinant N. europaea pheT in E. coli?

For optimal expression of recombinant N. europaea pheT in E. coli, researchers should consider the following protocol:

• Vector selection: pET-based expression vectors with T7 promoter systems have proven effective for pheT expression

• Host strain: BL21(DE3) or Rosetta(DE3) strains often yield higher expression levels due to codon optimization

• Induction conditions:

  • Temperature: 18-20°C after induction (reduces inclusion body formation)

  • IPTG concentration: 0.1-0.5 mM

  • Induction period: 16-18 hours

• Growth medium: LB or 2XYT supplemented with appropriate antibiotics

• Harvest time: Mid to late log phase (OD600 of 0.6-0.8) before induction

The lower induction temperature is particularly important as the N. europaea pheT protein may form inclusion bodies at higher temperatures. Co-expression with molecular chaperones such as GroEL/GroES may further improve soluble protein yield.

What purification strategies are most effective for obtaining active N. europaea pheT protein?

The purification of active N. europaea pheT protein requires a multi-step approach:

• Initial lysis:

  • Buffer composition: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5% glycerol, 1 mM DTT

  • Add protease inhibitor cocktail to prevent degradation

  • Gentle lysis via sonication (10-15 short pulses) or French press

• Initial capture:

  • Affinity chromatography using His-tag (IMAC) if recombinant protein has a histidine tag

  • Elution with 250-300 mM imidazole gradient

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose)

  • Buffer: 20 mM Tris-HCl pH 7.5, 50-500 mM NaCl gradient

• Final polishing:

  • Size exclusion chromatography (Superdex 200)

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT, 5% glycerol

• Activity preservation:

  • Store in small aliquots at -80°C with 10-15% glycerol

  • Avoid repeated freeze-thaw cycles

The purified protein should be assessed for activity using aminoacylation assays with tRNA(Phe) and radiolabeled phenylalanine to confirm functional integrity.

How can domain-specific mutations be introduced to study the functional regions of pheT?

To study functional regions of pheT through domain-specific mutations, researchers should employ the following methodologies:

This approach has been successful in identifying the role of the B2 domain as a secondary tRNA-binding site that contributes to editing by promoting the translocation of mischarged tRNA .

How does the N. europaea pheT subunit compare functionally with homologs from other bacterial species?

Comparative analysis of the N. europaea pheT subunit with homologs from other bacterial species reveals several important functional differences:

The N. europaea pheT subunit shows distinctive characteristics in its B2 domain function, which acts as a secondary tRNA-binding site that contributes to editing by promoting the translocation of mischarged tRNA to the editing site . This represents an evolutionary adaptation that may be related to the specific environmental niche of N. europaea as an ammonia-oxidizing chemolithoautotroph. Unlike some bacterial species that have evolved alternative mechanisms for preventing misaminoacylation, N. europaea relies on this post-transfer editing mechanism to maintain translational fidelity.

What is the impact of the genomic location of pheT on its expression and regulation in N. europaea?

The genomic location of pheT in N. europaea has significant implications for its expression and regulation:

• Co-regulation with energy metabolism genes:
  The positioning of pheT in the 15.5-kb intergenic spacer region between amo and hao gene clusters suggests potential co-regulation with genes involved in ammonia oxidation . This genomic arrangement may ensure coordinated expression of protein synthesis machinery with energy-generating pathways.

• Transcriptional organization:
  The pheT gene is part of a gene cluster that includes thrS, infC, rplT, and pheS, indicating a potential operon structure. This organization is similar to that found in many bacteria where translation-related genes are co-transcribed to ensure stoichiometric production of components.

• Response to environmental conditions:
  The expression levels of pheT likely fluctuate with ammonia availability and oxidation rates, as N. europaea is an obligate chemolithoautotroph that derives all its energy from ammonia oxidation . Under ammonia-limited conditions, translation machinery genes including pheT may be downregulated to conserve energy.

• Evolutionary conservation:
  The conserved genomic arrangement of pheT between N. europaea and Nitrosomonas sp. strain ENI-11 suggests functional importance. The maintenance of similar gene neighborhoods across different Nitrosomonas species indicates selective pressure to preserve this arrangement.

This genomic context may facilitate coordinated regulation of protein synthesis with the specialized metabolism of N. europaea, potentially contributing to the bacterium's adaptation to its ecological niche.

How can recombinant N. europaea pheT be utilized in genetic code expansion systems?

Recombinant N. europaea pheT could be engineered for genetic code expansion systems through the following approaches:

• Engineering substrate specificity:
  The beta subunit of PheRS contains domains involved in tRNA anticodon recognition and editing. By introducing targeted mutations to these domains, researchers could potentially create variants with altered specificity that accept non-canonical amino acids (ncAAs).

• Development of orthogonal synthetase-tRNA pairs:
  The N. europaea PheRS system could be engineered to function orthogonally to the host's translational machinery. This would involve:

  • Modifying the tRNA(Phe) anticodon to recognize amber stop codons

  • Altering the synthetase to recognize only the modified tRNA

  • Engineering the amino acid binding pocket to accommodate ncAAs

• Selection strategies for optimized variants:
  Similar to the approach used with pyrrolysyl-tRNA synthetase (PylRS) systems , researchers could employ:

  • Positive selection using a selectable marker (e.g., chloramphenicol acetyl transferase) containing an amber codon

  • Negative selection to eliminate variants that incorporate natural amino acids

  • Screening in the presence of the desired ncAA

• Potential advantages of N. europaea PheRS:

  • The unique B2 domain could provide additional engineering sites

  • The editing domain could be modified to prevent hydrolysis of ncAA-tRNA adducts

  • The adaptation to extreme environments might confer stability advantages

This application represents an advanced research direction that could expand the toolkit for genetic code expansion beyond the commonly used pyrrolysyl and tyrosyl systems.

What are common challenges in working with recombinant N. europaea pheT and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant N. europaea pheT:

• Low solubility and inclusion body formation:

  • Solution: Use lower induction temperatures (16-18°C), reduced IPTG concentrations (0.1-0.2 mM), and solubility-enhancing fusion tags (SUMO, MBP)

  • Alternative approach: Optimize refolding protocols from inclusion bodies using step-wise dialysis with decreasing urea/guanidine concentrations

• Incomplete complex formation with alpha subunit:

  • Solution: Co-express pheS and pheT using a bicistronic construct to ensure proper stoichiometry

  • Verification method: Size exclusion chromatography to confirm (αβ)₂ tetramer formation

• Reduced editing activity:

  • Solution: Ensure the integrity of the B2 domain during purification, as this domain contributes to editing by promoting the translocation of mischarged tRNA

  • Assessment method: Compare Tyr-tRNA(Phe) hydrolysis rates with published values

• Inconsistent aminoacylation activity:

  • Solution: Add zinc ions (25-50 μM ZnCl₂) to reaction buffers, as zinc is often required for proper folding and activity

  • Control test: Include commercially available E. coli PheRS as a positive control

• Protein degradation during purification:

  • Solution: Include protease inhibitors throughout purification and maintain low temperatures

  • Alternative approach: Engineer constructs with stabilizing mutations based on molecular dynamics simulations

Troubleshooting these issues requires systematic optimization of expression and purification conditions, along with careful activity assays to verify functional integrity.

How can researchers analyze and interpret contradictory data regarding pheT domain functions?

When faced with contradictory data regarding pheT domain functions, researchers should employ the following analytical approach:

• Evaluate methodological differences:

  • Compare experimental conditions (pH, temperature, salt concentration)

  • Assess protein construct boundaries (full-length vs. truncated versions)

  • Review the source of tRNA (in vitro transcribed vs. native)

  • Examine the techniques used to measure activity (steady-state vs. pre-steady-state kinetics)

• Perform integrative data analysis:

  • Combine structural information with functional data

  • Use multiple complementary techniques to verify results (e.g., both gel-based assays and fluorescence-based methods)

  • Apply statistical methods to determine significance of observed differences

• Address specific B2 domain contradictions:

  • Structural studies indicate the B2 domain is distant from bound tRNA(Phe), yet deletion affects function

  • Resolution: The domain may act as a secondary binding site that facilitates tRNA movement between aminoacylation and editing sites

  • Test this hypothesis with single-molecule FRET to track tRNA movement

• Reconcile in vitro versus in vivo discrepancies:

  • Compare results from purified protein studies with those from cellular complementation

  • Consider potential interaction partners present in vivo but absent in purified systems

  • Examine the effects of cellular conditions (molecular crowding, ion concentrations)

• Consider evolutionary context:

  • Compare with homologous systems from other organisms

  • Analyze conservation patterns of disputed domains

  • Evaluate the relationship between genomic context and function in N. europaea

This systematic approach can help researchers resolve contradictions and develop a more comprehensive understanding of pheT domain functions.

What novel applications of recombinant N. europaea pheT might emerge from current research trends?

Current research trends suggest several promising novel applications for recombinant N. europaea pheT:

• Biosensors for environmental monitoring:

  • The editing function of pheT could be engineered to create sensors for specific environmental contaminants

  • Modified pheT proteins could generate fluorescent signals upon binding to target molecules

  • These biosensors could be particularly useful for monitoring environments where N. europaea naturally thrives, such as wastewater treatment systems

• Synthetic biology tools:

  • The unique properties of the B2 domain could be harnessed to create modular RNA-binding scaffolds

  • These scaffolds could be used to organize metabolic pathways through RNA-protein interactions

  • The editing domain could be repurposed to create selective RNA degradation systems

• Protein evolution platforms:

  • The natural editing function could be adapted to create selection systems for protein engineering

  • By linking editing activity to cell survival, researchers could develop new directed evolution methods

  • This could enable the discovery of novel catalysts with applications in bioremediation

• Drug discovery targets:

  • The unique features of N. europaea pheT compared to human PheRS could be exploited for developing selective inhibitors

  • Such inhibitors might have applications against related pathogenic bacteria

  • Structure-based drug design could focus on the B2 domain and its interactions

• Bioremediation of nitrogenous compounds:

  • Engineered N. europaea expressing modified pheT could potentially incorporate toxic amino acid analogs into proteins

  • This could enhance the organism's ability to process certain environmental contaminants

  • Applications could include treatment of agricultural runoff or industrial waste

These emerging applications highlight the potential of N. europaea pheT beyond its natural role in protein synthesis.

What are the most promising approaches for studying the structural dynamics of pheT during the editing process?

The most promising approaches for studying the structural dynamics of pheT during the editing process include:

• Time-resolved cryo-electron microscopy (cryo-EM):

  • Capture different conformational states during the editing process

  • Use rapid freezing techniques to trap intermediate states

  • Combine with computational modeling to create dynamic visualizations

• Single-molecule Förster resonance energy transfer (smFRET):

  • Attach fluorescent probes to specific domains of pheT and its tRNA substrate

  • Monitor real-time movement of the B2 domain during tRNA translocation

  • Correlate FRET efficiency changes with biochemical steps in the editing pathway

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Probe solvent accessibility changes during different stages of editing

  • Identify regions that undergo conformational changes

  • Map interaction interfaces between pheT domains and tRNA

• Molecular dynamics (MD) simulations:

  • Model the complete (αβ)₂ tetramer with bound tRNA

  • Simulate the translocation of mischarged tRNA from synthetic to editing sites

  • Predict the role of the B2 domain in facilitating this movement

• Site-specific crosslinking coupled with mass spectrometry:

  • Introduce photo-activatable crosslinkers at specific positions

  • Capture transient interactions during the editing process

  • Identify precise contact points between pheT domains and tRNA

• Nuclear magnetic resonance (NMR) spectroscopy:

  • Study domain dynamics of isolated B2 domain

  • Characterize its interaction with tRNA fragments

  • Monitor chemical shift perturbations upon binding

These complementary approaches would provide unprecedented insights into how the B2 domain of pheT contributes to editing by promoting the translocation of mischarged tRNA to the editing site of PheRS.

How might understanding N. europaea pheT contribute to our knowledge of protein synthesis evolution in specialized bacterial niches?

Understanding N. europaea pheT could provide valuable insights into protein synthesis evolution in specialized bacterial niches through several avenues:

• Adaptation to energy-limited environments:

  • N. europaea is an obligate chemolithoautotroph that derives all its energy from ammonia oxidation

  • The efficiency of its translation machinery, including PheRS, may reflect adaptations to this energy-constrained lifestyle

  • Comparative studies with heterotrophic bacteria could reveal optimization strategies for protein synthesis under energy limitation

• Co-evolution with metabolic specialization:

  • The genomic proximity of pheT to ammonia oxidation genes suggests potential co-evolutionary relationships

  • Analysis of selection pressures on different PheRS domains could reveal how translation machinery adapts to specialized metabolic pathways

  • This might explain why the genomic arrangement is conserved between different Nitrosomonas species

• Fidelity mechanisms in extreme environments:

  • The editing function of the B2 domain may represent a specific adaptation to maintain translational fidelity under the unique physiological conditions of ammonia-oxidizing bacteria

  • Comparing editing mechanisms across bacteria from different ecological niches could reveal environment-specific solutions to the challenge of translational accuracy

• Horizontal gene transfer and synthetase evolution:

  • Analysis of pheT sequence conservation and phylogeny could reveal instances of horizontal gene transfer

  • This would help understand how aminoacyl-tRNA synthetases evolve in specialized bacterial communities

  • Potential implications for the spread of antibiotic resistance genes that may piggyback on synthetase gene transfers

• Evolutionary trade-offs between efficiency and accuracy:

  • The specific characteristics of N. europaea pheT may represent evolutionary trade-offs between translational speed and accuracy

  • This could inform our understanding of how protein synthesis adapts to different selective pressures in specialized ecological niches

This research direction would contribute to the broader understanding of how core cellular processes adapt to specialized ecological contexts and metabolic strategies.