Recombinant Enterococcus faecalis tRNA (Ile)-lysidine synthase (tilS)

What is tRNA (Ile)-lysidine synthase (tilS) and what is its biological significance?

tRNA (Ile)-lysidine synthase (tilS) is an essential enzyme in bacteria that catalyzes the ATP-dependent modification of cytidine at the wobble position (position 34) of the tRNA^Ile2 anticodon to lysidine. This posttranscriptional modification is critical for bacterial survival as it converts the codon recognition specificity from AUG to AUA and switches the amino acid charging specificity from methionine to isoleucine .

The lysidine modification serves as both a positive determinant for isoleucyl-tRNA synthetase (IleRS) and a negative determinant for methionyl-tRNA synthetase (MetRS), thereby ensuring translational fidelity. The gene encoding this enzyme, tilS (formerly yacA), has been identified in both Escherichia coli and Bacillus subtilis, and is nearly universally conserved across eubacteria, including numerous human pathogens . Importantly, the absence of tilS homologs in mammalian systems makes it a potentially attractive antibacterial target.

What experimental evidence demonstrates that lysidine modification is necessary for correct tRNA function?

Complementary evidence shows that a C34G mutation in tRNA^Ile2 prevents tilS-catalyzed modification. When tested as an inhibitor rather than a substrate, this mutated tRNA exhibits potent inhibition with an IC₅₀ of 85 nM, confirming the critical importance of cytidine at position 34 for enzymatic recognition while simultaneously demonstrating that other structural features of the tRNA are crucial for enzyme binding .

How does the tilS reaction mechanism differ from other tRNA modification enzymes?

Unlike many tRNA modification enzymes that perform simple chemical transformations, tilS catalyzes a complex reaction involving complete replacement of the C2 carbonyl oxygen of cytidine with a lysyl group. The reaction proceeds through an ATP-dependent activation step followed by nucleophilic attack by the ε-amino group of lysine.

What makes tilS particularly unique is its catalytic flexibility. Studies have revealed that in addition to lysine, tilS can utilize various lysine analogs and even simple alkyl amines as alternative substrates . This flexibility distinguishes tilS from most other tRNA modification enzymes that typically display high substrate specificity. The enzyme appears to primarily recognize the tRNA structure rather than having stringent requirements for the lysine substrate, which has important implications for both mechanistic understanding and potential inhibitor design.

What are the optimal conditions for expressing and purifying recombinant tilS?

For efficient expression and purification of recombinant tilS, the following methodological approach has proven effective:

• Expression system: Clone the tilS gene into the pET28a vector with an N-terminal His₆-tag for expression in E. coli BL21(DE3) .

• Induction conditions: Grow cultures to mid-log phase (OD₆₀₀ = 0.6) at 37°C, then induce with 1 mM IPTG for 3 hours .

• Cell lysis: Resuspend harvested cells in buffer containing 50 mM HEPES (pH 7.6), 10 mM MgCl₂, 500 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM β-mercaptoethanol .

• Purification sequence:

  • Initial purification via Ni²⁺-NTA affinity chromatography

  • Ammonium sulfate precipitation (65% saturation)

  • Resuspension and dialysis against 50 mM HEPES (pH 7.6), 10 mM MgCl₂

  • Storage with 10% glycerol at -80°C

This protocol typically yields highly pure and active enzyme suitable for kinetic and substrate specificity studies.

How can researchers prepare suitable tRNA^Ile2 substrates for in vitro studies?

Preparation of functional tRNA^Ile2 substrates for tilS studies requires careful attention to structural integrity. The recommended approach involves in vitro transcription following these steps:

• Template construction: Create a DNA template by placing the tRNA gene behind a T7 promoter using overlapping oligonucleotides .

• Primer design: Use forward primer (5′-GTGGTTAGAGCAGGCGACTGATAATCGCTTGGTCGCT-3′) and reverse primer (5′-CCAGCGACCAAGCGATTATCAGTCGCCTGCTCTAACCAC-3′) for PCR amplification .

• Verification: Confirm template sequence before proceeding to transcription.

• Transcription reaction: Use purified DNA template with T7 RNA polymerase under standard conditions.

• Purification: Employ gel electrophoresis to isolate the correctly sized tRNA transcript.

For studying C34G-mutated tRNA^Ile2, prepare the template by site-directed mutagenesis of the wild-type construct. This mutant is valuable as a control or inhibitor since it binds tilS but is not modified .

What assay systems provide quantitative measurement of tilS activity?

Several complementary assay systems can be employed to quantitatively measure tilS activity:

• Radiometric assay: The standard assay utilizes [³H]lysine incorporation into tRNA, with the following components:

  • 50 mM TAPS buffer (pH 8.5)

  • 3 mM MgCl₂

  • 0.01% Tween 20

  • 0.5 mM TCEP

  • 0.1 mg/ml bovine serum albumin

  • 20 nM E. coli tRNA^Ile2

  • 5 μM ATP

  • 110 nM [³H]lysine (91 Ci/mmol)

  • 10 nM E. coli tilS enzyme

  Reactions are incubated for 2 hours at room temperature, terminated by the addition of SPA beads in citrate buffer, and radioactivity measured by scintillation counting .

• Gel electrophoresis: Modified tRNAs can be separated on 15% polyacrylamide Tris borate-EDTA gels containing 8 M urea, providing visual confirmation of modification .

• Mass spectrometry: For definitive identification of tRNA modifications, especially with alternative substrates .

• Coupled aminoacylation assay: Measures the functional consequence of tilS modification by assessing subsequent charging by IleRS .

How does tilS accommodate different nucleotide substrates, and what are the kinetic implications?

tilS exhibits notable flexibility in accommodating nucleotide substrate analogs, though with varying kinetic efficiency. The table below summarizes the kinetic parameters for different nucleotide substrates:

This data reveals that while tilS can utilize various ATP analogs, the catalytic efficiency (Vₘₐₓ/Kₘ) is significantly reduced compared to ATP itself . The 7-Deazaadenosine-5′-triphosphate analog retains a Kₘ value similar to ATP but with approximately 12-fold lower relative catalytic efficiency, suggesting that modification at the 7-position is less disruptive than modifications at other positions.

The substantial increase in Kₘ values for 8-Azidoadenosine-5′-triphosphate and N6-Methyladenosine-5′-triphosphate indicates reduced binding affinity, likely due to steric hindrance or altered hydrogen bonding capabilities . Researchers investigating the ATP-binding mechanism of tilS should consider these differential effects when designing experiments.

What is the range of lysine analogs that can serve as alternative substrates for tilS?

A surprising feature of tilS is its ability to accept a wide range of lysine analogs and even simple alkyl amines as alternative substrates. Experimental evidence confirms that many compounds containing a primary amine can be incorporated into tRNA^Ile2 by tilS. The following analogs have been demonstrated to function as substrates:

• Lysine derivatives with modified carboxyl groups

• Lysine derivatives with modified α-amino groups

• Shorter chain diamines (e.g., ornithine, 2,4-diaminobutyric acid)

• Simple alkyl amines

How do researchers confirm the incorporation of alternative substrates into tRNA?

Confirming the incorporation of alternative substrates into tRNA by tilS requires a multi-faceted analytical approach:

• Indirect assessment: Measure the depletion of unmodified tRNA substrate by diluting an aliquot of the reaction mixture into a standard assay containing tritiated lysine. Complete disappearance of the signal indicates full conversion of the original tRNA substrate .

• Gel electrophoresis: Modified tRNAs often display altered mobility on polyacrylamide gels containing urea, providing visual confirmation of modification. As shown in research studies, tRNAs modified with different lysine analogs can be distinguished from unmodified tRNA and from each other .

• Mass spectrometry: For definitive identification, mass spectrometry provides precise molecular weight determination of the modified nucleoside. This technique can distinguish between various modifications based on the mass shift relative to unmodified cytidine .

• Functional analysis: Assess whether the alternatively modified tRNAs can serve as substrates for aminoacylation by IleRS and/or MetRS. This confirms whether the modifications fulfill the biological role of converting tRNA^Ile2 specificity .

How do different tRNA modifications affect recognition by aminoacyl-tRNA synthetases?

The modification of tRNA^Ile2 by tilS fundamentally alters its recognition by aminoacyl-tRNA synthetases. Research has demonstrated that all modifications catalyzed by tilS—whether with lysine or various analogs—effectively create:

• Negative determinants for MetRS: Preventing mischarging with methionine

• Positive determinants for IleRS: Enabling proper charging with isoleucine

For researchers, these findings highlight the importance of considering the source organism when studying tRNA-synthetase interactions. Experiments designed to investigate modification requirements should include comparative analyses with enzymes from multiple bacterial species.

What is the potential of tilS as a target for novel antibacterial development?

Several characteristics make tilS an attractive potential target for novel antibacterial development:

• Essentiality: The tilS gene has been demonstrated to be essential for viability in bacteria such as B. subtilis .

• Bacterial specificity: There is no mammalian counterpart to tilS, as eukaryotes use tRNA^Ile species with either inosine or modified uridine at the wobble position to recognize isoleucine codons .

• Conservation: TilS homologs are nearly universally present in eubacteria, including numerous human pathogens, suggesting that inhibitors could potentially have broad-spectrum activity .

• Mechanistic understanding: High-resolution protein structures of bacterial tilS are available, and a straightforward reaction mechanism has been proposed, facilitating structure-based inhibitor design .

• Functional consequence of inhibition: Partial inactivation of tilS in E. coli leads to an AUA codon-dependent translational defect, suggesting that inhibitors could specifically disrupt bacterial protein synthesis .

How can researchers leverage the catalytic flexibility of tilS for experimental applications?

The remarkable catalytic flexibility of tilS offers several innovative experimental applications:

• Probing tRNA-synthetase interactions: By generating tRNA^Ile2 with various modified bases at position 34, researchers can systematically investigate the structural requirements for recognition by different aminoacyl-tRNA synthetases .

• Creating tRNA with novel properties: The ability to incorporate unnatural amino acid derivatives into tRNA opens possibilities for engineering tRNAs with altered decoding properties or chemical functionalities.

• Mechanistic studies: The tolerance for diverse substrates enables detailed structure-activity relationship studies of the tilS reaction mechanism.

• Development of tilS inhibition assays: Understanding the range of acceptable substrates informs the design of high-throughput screening assays for identifying potential inhibitors.

Researchers should note that the differential effects of various modifications on subsequent tRNA function provide a valuable tool for dissecting the precise structural requirements for tRNA recognition by different cellular components.

What are common challenges in expressing and purifying active recombinant tilS?

Researchers frequently encounter several challenges when working with recombinant tilS:

• Solubility issues: TilS can form inclusion bodies when overexpressed. To mitigate this, consider:

  • Lowering induction temperature to 18-25°C

  • Reducing IPTG concentration to 0.1-0.5 mM

  • Including solubility enhancers such as sorbitol or glycerol in growth media

• Activity loss during purification: TilS activity can be sensitive to oxidation. Ensure all buffers contain reducing agents such as TCEP (0.5 mM) or β-mercaptoethanol (5 mM) .

• Stability concerns: Purified tilS may lose activity during storage. Addition of 10% glycerol and storage at -80°C rather than -20°C helps maintain enzymatic activity .

• Batch variability: Different preparations may show variable specific activity. Always include a standardized activity assay to normalize enzyme concentrations across different preparations.

How should researchers interpret and troubleshoot anomalous results in tilS assays?

When investigating tilS activity, several types of anomalous results may be encountered:

• Apparent inhibition that is actually substrate competition: When testing potential inhibitors containing primary amines, apparent inhibition may actually represent alternative substrate utilization. To distinguish:

  • Test whether tRNA remains a substrate for standard tilS assays after treatment

  • Analyze modified tRNA by gel electrophoresis or mass spectrometry

• Biphasic kinetics: Some substrates may show complex kinetic patterns. This could reflect:

  • Multiple binding modes

  • Substrate inhibition at high concentrations

  • Product inhibition

• Inconsistent results with different tRNA preparations: In vitro transcribed tRNA may lack the full activity of naturally modified tRNA. Ensure consistent preparation methods and consider including control reactions with commercial tRNA preparations when available.

• Species-specific differences: Remember that tilS and IleRS from different bacterial species may show different substrate specificities. When results differ from published data, consider whether the source organism differs from previous studies .

What are promising approaches for developing selective inhibitors of tilS?

Based on current understanding of tilS structure and mechanism, several approaches show promise for developing selective inhibitors:

• ATP-competitive inhibitors: Given the well-defined ATP binding pocket, designing nucleotide analogs that compete with ATP but cannot be utilized in the reaction represents a viable strategy .

• Bisubstrate analogs: Compounds that simultaneously mimic portions of both ATP and lysine could achieve higher potency and selectivity.

• Allosteric inhibitors: Targeting sites away from the active site could avoid the challenge posed by the enzyme's broad substrate acceptance.

• tRNA-competitive inhibitors: As demonstrated by the potent inhibition of C34G-mutated tRNA^Ile2 (IC₅₀ of 85 nM), developing small molecules that mimic critical tRNA binding determinants represents a promising approach .

• Transition state analogs: Designing compounds that mimic the proposed reaction transition state could yield highly potent inhibitors.

The design strategy should account for the enzyme's catalytic flexibility while exploiting the essential nature of the reaction for bacterial survival.

How might understanding tilS evolution inform bacterial adaptation studies?

The evolutionary aspects of tilS offer rich territory for research into bacterial adaptation:

• Comparative genomics: Analyzing tilS sequence conservation and divergence across bacterial phyla can reveal evolutionary pressures and adaptation mechanisms.

• Co-evolution with tRNA^Ile2: Investigating how tilS and its tRNA substrate have co-evolved provides insight into the development of bacterial translational systems.

• Species-specific differences in substrate specificity: The observed differences in substrate tolerance between E. coli and B. subtilis IleRS suggest adaptation to different environmental niches .

• Horizontal gene transfer: Assessing whether tilS genes show evidence of horizontal transfer could inform understanding of bacterial evolution.

• Antibiotic resistance mechanisms: Studying potential resistance mechanisms against tilS inhibitors before they are developed could guide preemptive strategies in antibacterial design.

These evolutionary studies have implications not only for basic research but also for anticipating bacterial responses to potential therapeutic interventions targeting tilS.