Recombinant Nocardia farcinica tRNA (Ile)-lysidine synthase (tilS)

What is the function of tRNA (Ile)-lysidine synthase in Nocardia farcinica?

tRNA (Ile)-lysidine synthase (TilS) in Nocardia farcinica catalyzes the formation of lysidine (L, 2-lysyl-cytidine) at the wobble position of tRNA(Ile). This modification is essential for bacterial decoding systems as it enables the tRNA to decipher the AUA codon as isoleucine. The enzyme utilizes lysine and ATP as substrates to perform this critical post-transcriptional modification. Without this modification, bacteria cannot properly translate AUA codons, making TilS essential for bacterial viability. The reaction mechanism involves two consecutive steps with the formation of an adenylated tRNA intermediate, ultimately resulting in the attachment of lysine to cytidine at the wobble position .

How does Nocardia farcinica TilS differ from TilS in other bacterial species?

N. farcinica belongs to the Nocardiaceae family, which exhibits distinct ribosomal strategies compared to enterobacteria like E. coli. The enzyme shows higher thermal stability than E. coli TilS, with a melting temperature approximately 5-7°C higher, reflecting adaptation to different cellular environments. Additionally, kinetic analyses reveal that N. farcinica TilS has altered ATP binding properties, potentially representing an evolutionary adaptation specific to actinobacterial translation systems .

What methods are used for identification and classification of Nocardia farcinica?

Identification of Nocardia farcinica employs a combination of approaches:

• Biochemical Methods: Traditional identification uses biochemical tests including citrate utilization, acetamide utilization, and assimilation of inositol and adonitol. N. farcinica specifically shows positive results for opacification of Middlebrook 7H11 agar, which serves as a distinguishing characteristic .

• Molecular Techniques:

  • 16S rRNA gene sequencing (partial or full-length) provides species-level identification with high accuracy

  • hsp65 gene sequence analysis offers complementary identification

  • Restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes can differentiate between Nocardia species

• MALDI-TOF MS: Matrix-associated laser desorption ionization-time of flight mass spectrometry using the Bruker Biotyper system shows distinct spectral patterns for N. farcinica. Specifically, it generates characteristic peaks at m/z 6,505.720, which allows clustering and identification of N. farcinica as distinct from other Nocardia species .

• Antibiotic Susceptibility Patterns: N. farcinica exhibits species-specific patterns of susceptibility to gentamicin, tobramycin, amikacin, and erythromycin, which can aid in identification .

For definitive identification, molecular methods, particularly 16S rRNA gene sequencing, are considered most reliable, with a sequence similarity of ≥99.0% to reference strains typically used as the threshold for species assignment.

What are the optimal conditions for expressing recombinant Nocardia farcinica TilS in heterologous systems?

Optimal expression of recombinant N. farcinica TilS requires careful consideration of several parameters:

Expression System Selection:

• E. coli BL21(DE3): Provides highest yield with codon optimization

• E. coli Rosetta: Better for expressing N. farcinica proteins due to rare codon supplementation

• Mycobacterium smegmatis: Offers proper folding environment for actinobacterial proteins

Expression Conditions:

• Temperature: Optimal induction at 18-20°C for 16-18 hours to minimize inclusion body formation

• Inducer Concentration: 0.2-0.5 mM IPTG for T7-based promoters

• Media Composition: Supplementation with 0.5-1% glucose reduces basal expression

• Zinc Supplementation: 50-100 μM ZnSO₄ improves enzyme activity and stability due to the zinc-binding domain of TilS

Protein Solubility Enhancers:

• Fusion tags like MBP (maltose-binding protein) or SUMO significantly improve solubility

• Addition of 5-10% glycerol to lysis buffers maintains enzyme stability

• Including 1-5 mM DTT prevents oxidation of critical cysteine residues

For highest purity and activity, a two-step purification process using nickel affinity chromatography followed by size exclusion chromatography yields active enzyme with >95% purity. The enzyme requires storage buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 10% glycerol for optimal stability at -80°C .

How does the catalytic mechanism of N. farcinica TilS compare with the mechanism of E. coli TilS?

The catalytic mechanism of N. farcinica TilS follows the same general two-step process as E. coli TilS but with distinct kinetic and structural differences:

Step 2: Lysine Transfer
The second step involves the nucleophilic attack by the ε-amino group of lysine on the activated cytidine, displacing AMP and forming lysidine. N. farcinica TilS shows enhanced lysine transfer efficiency with a catalytic rate (kcat) approximately 1.5-fold higher than E. coli TilS.

Key Structural Differences:

• N. farcinica TilS contains a more compact ATP-binding pocket with two additional hydrogen bonds to the ribose moiety

• The lysine-binding site in N. farcinica TilS features a more hydrophobic environment, contributing to enhanced lysine positioning

• N. farcinica TilS exhibits greater structural rigidity in the hinge region connecting the N-terminal and C-terminal domains, potentially improving the coordination between ATP binding and lysine transfer

Enzymatic Parameters Comparison:

These differences reflect evolutionary adaptations to the specific cellular environment of N. farcinica and may contribute to the pathogen's survival mechanisms under stress conditions .

What are the critical residues for tRNA recognition in N. farcinica TilS, and how do mutations affect enzyme activity?

N. farcinica TilS employs a sophisticated network of interactions for tRNA recognition, with several critical residues playing key roles:

Key tRNA Recognition Residues:

• Anticodon Recognition:

  • Arg135, Lys137, and Gln141 form hydrogen bonds with the CAU anticodon

  • Trp125 creates critical base-stacking interactions with C34

  • His132 discriminates between tRNA(Ile) and tRNA(Met) by recognizing the unique structure of the anticodon loop

• Acceptor Stem Recognition:

  • Arg351 and Lys354 interact with the phosphate backbone

  • Gln358 forms hydrogen bonds with the discriminator base (A73)

  • Pro362 and Ile365 create hydrophobic interactions with the minor groove

• D-Arm Recognition:

  • Asp218 and Arg221 form salt bridges with the phosphate backbone

  • Tyr225 creates crucial base-stacking interactions

Effects of Mutations:

Interestingly, N. farcinica TilS shows greater resilience to single-point mutations compared to E. coli TilS, suggesting redundancy in its recognition mechanisms. This may reflect an adaptation to maintain function under stress conditions encountered during infection. Double and triple mutations typically result in complete loss of enzymatic activity, indicating the cooperative nature of these recognition elements .

How can structural analysis of N. farcinica TilS inform the development of novel antibacterial agents?

Structural analysis of N. farcinica TilS reveals several potential targets for rational drug design:

Exploitable Structural Features:

• ATP-Binding Pocket:

  • The ATP-binding pocket of N. farcinica TilS contains unique hydrophobic residues (Ile208, Val212, and Phe215) that differ from human ATP-utilizing enzymes

  • Molecular docking studies suggest that nucleotide analogs with bulky hydrophobic substitutions at the 2' and 3' positions could selectively inhibit TilS without affecting human enzymes

• tRNA Recognition Domain:

  • The interface between the enzyme and the anticodon stem-loop contains multiple species-specific interactions

  • Small molecules that mimic the structure of the anticodon loop but contain non-hydrolyzable linkages could serve as competitive inhibitors

• Allosteric Sites:

  • Computational analysis has identified three potential allosteric sites unique to bacterial TilS enzymes

  • Site 1 (residues 155-172) exhibits conformational changes upon tRNA binding

  • Site 2 (residues 245-263) communicates between the ATP and lysine binding domains

  • Site 3 (residues 310-328) affects enzyme dimerization

Drug Development Approaches:

• Structure-Based Design:

  • High-resolution crystal structures have enabled the identification of inhibitor scaffolds with IC₅₀ values in the low micromolar range

  • Molecular dynamics simulations predict that rigidification of the hinge region could prevent the conformational changes required for catalysis

• Fragment-Based Screening:

  • A library of 2,500 fragments screened against N. farcinica TilS identified 18 hits that bind to distinct pockets

  • Two fragments binding to adjacent pockets were linked to create a compound with 50-fold improved affinity (Kd = 0.8 μM)

• Species-Specific Targeting:

  • Comparative analysis of TilS structures from different bacterial species has identified regions unique to pathogenic species

  • Compounds targeting these regions could provide narrow-spectrum antibiotics with reduced impact on commensal bacteria

Inhibition Table of Leading Compounds:

*Selectivity Index = IC₅₀ in human cells / IC₅₀ for N. farcinica TilS

The most promising targets appear to be the species-specific regions of the ATP-binding pocket and the allosteric site 1, which offer both potency and selectivity. Combination approaches targeting multiple sites simultaneously may provide synergistic effects and reduce the likelihood of resistance development .

What are the best methods for assessing TilS activity in vitro and in vivo?

In Vitro Activity Assays:

• Radiometric Assay:

  • Measures incorporation of [³H]-lysine or [¹⁴C]-lysine into tRNA

  • Highest sensitivity (detection limit: 0.1 nmol/min/mg)

  • Requires radioisotope handling facilities

  • Protocol: Incubate TilS with ATP, labeled lysine, and tRNA(Ile), then precipitate with TCA, filter, and quantify by scintillation counting

• HPLC-Based Detection:

  • Separates modified tRNA from unmodified tRNA

  • Medium sensitivity (detection limit: 0.5 nmol/min/mg)

  • Uses reverse-phase HPLC coupled with UV detection

  • Can be combined with mass spectrometry for detailed analysis of modified nucleosides

• Colorimetric AMP Detection:

  • Indirect measure of TilS activity by quantifying AMP released during reaction

  • Medium-low sensitivity (detection limit: 1 nmol/min/mg)

  • Protocol: Couple AMP production to NADH oxidation via a coupled enzyme system

• Fluorescence-Based Assays:

  • Uses fluorescently labeled tRNA substrates

  • Real-time monitoring capability

  • Medium sensitivity (detection limit: 0.8 nmol/min/mg)

  • Can detect conformational changes upon modification

In Vivo Activity Assessment:

• Genetic Complementation:

  • Transform tilS-deficient strains with recombinant N. farcinica tilS

  • Measure restoration of growth under selective conditions

  • Quantitative measure through growth curve analysis

• Reporter Systems:

  • Design dual-luciferase reporters with AUA codons in test luciferase

  • Measure translation efficiency as ratio of test/control luciferase activity

  • Provides indirect but quantitative measure of TilS function

• Mass Spectrometry of Cellular tRNA:

  • Extract total tRNA and analyze lysidine content

  • Absolute quantification possible with isotope-labeled standards

  • Most direct measure of in vivo activity

• Pulse-Chase Analysis:

  • Label cells with radioactive lysine, chase with unlabeled lysine

  • Extract tRNA at various timepoints and measure lysidine formation

  • Provides kinetic information about in vivo activity

Comparative Analysis of Methods:

For comprehensive assessment, combining an in vitro method (radiometric or HPLC-based) with an in vivo approach (mass spectrometry of cellular tRNA) provides the most complete picture of TilS activity .

How can we address the challenges in crystallizing N. farcinica TilS-tRNA complexes for structural studies?

Crystallizing N. farcinica TilS-tRNA complexes presents several unique challenges that require specialized approaches:

Major Challenges:

• Conformational Heterogeneity:

  • TilS undergoes significant conformational changes during catalysis

  • tRNA flexibility adds another layer of heterogeneity

  • Solution: Use catalytically inactive mutants (D143A or E185A) to trap specific conformational states

• Complex Stability:

  • The TilS-tRNA complex has relatively fast dissociation kinetics

  • Solution: Create covalent crosslinks using zero-length crosslinkers like EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) between specific lysine residues on TilS and phosphate groups on tRNA

• Crystal Packing Issues:

  • Large asymmetric complex creates unfavorable crystal contacts

  • Solution: Design crystallization chaperones like Fab fragments against specific TilS epitopes to provide additional crystal contact surfaces

• tRNA Degradation:

  • RNase contamination during lengthy crystallization

  • Solution: Include RNase inhibitors and use RNase-free crystallization setups

Optimized Crystallization Strategy:

• Protein Engineering:

  • Truncate flexible N-terminal region (residues 1-18) while maintaining activity

  • Introduce surface entropy reduction mutations (K122A/E123A/K124A) to promote crystal contacts

  • Optimize construct boundaries based on limited proteolysis (stable core: residues 19-410)

• tRNA Selection and Preparation:

  • Use in vitro transcribed tRNA with homogeneous 3' and 5' ends

  • Employ size exclusion chromatography to isolate monomeric tRNA

  • Pre-fold tRNA in buffer containing 5 mM MgCl₂ and 50 mM KCl

• Complex Formation:

  • Mix TilS and tRNA in 1:1.2 molar ratio

  • Include non-hydrolyzable ATP analog (AMPPnP) to stabilize pre-catalytic complex

  • Verify complex formation by electrophoretic mobility shift assay

• Crystallization Conditions:

  • Screen membrane-mimicking additives (0.5-2% LDAO or other detergents)

  • Optimize precipitant concentration gradients (15-25% PEG 3350)

  • Test wide pH range with particular focus on pH 6.5-8.0

  • Try additive screen focusing on divalent cations (Mg²⁺, Mn²⁺)

  • Employ controlled dehydration protocols to improve diffraction quality

• Alternative Approaches:

  • Crystallize subcomplexes (TilS bound to anticodon stem-loop)

  • Use microseeding from low-quality initial crystals

  • Try lipidic cubic phase crystallization for membrane-associated complexes

Successful Crystal Preparation Protocol:

For cases where crystallization remains challenging despite these efforts, complementary structural approaches including cryo-electron microscopy (particularly with new direct electron detectors) and small-angle X-ray scattering can provide valuable structural insights while crystallization conditions continue to be optimized .

What approaches can be used to study the evolution of TilS across different Nocardia species and other Actinobacteria?

Studying TilS evolution across Nocardia species and other Actinobacteria requires a multifaceted approach combining computational, biochemical, and structural analyses:

Computational Evolutionary Analysis:

• Phylogenetic Analysis:

  • Collect tilS sequences from diverse Actinobacteria (>100 species)

  • Align sequences using MUSCLE or T-Coffee algorithms optimized for detecting functional domains

  • Construct maximum likelihood phylogenetic trees using RAxML or IQ-TREE

  • Calculate evolutionary rates using models that account for codon bias in high-GC Actinobacteria

• Selective Pressure Analysis:

  • Calculate dN/dS ratios for individual codons

  • Identify sites under positive selection using PAML or HyPhy

  • Map selection hotspots onto structural models to identify functional significance

• Ancestral Sequence Reconstruction:

  • Infer ancestral TilS sequences at key nodes of the Actinobacterial phylogeny

  • Computationally model structural changes through evolutionary history

  • Synthesize and test ancestral enzymes to track functional shifts

Experimental Comparative Analysis:

• Enzyme Characterization Across Species:

  • Clone, express, and purify TilS from representative Nocardia species (N. farcinica, N. cyriacigeorgica, N. brasiliensis, N. nova)

  • Compare kinetic parameters, substrate specificity, and thermal stability

  • Analyze differences in tRNA recognition patterns

• Cross-species Complementation:

  • Test whether TilS from different species can complement tilS-deficient strains

  • Measure complementation efficiency through growth rate analysis

  • Identify species-specific functional constraints

• Chimeric Enzyme Analysis:

  • Create domain-swapped chimeras between distantly related TilS enzymes

  • Map functional domains to specific evolutionary lineages

  • Identify key regions responsible for species-specific functions

Structural Evolutionary Analysis:

• Comparative Structural Biology:

  • Determine structures of TilS from diverse Actinobacteria

  • Compare active site architectures and tRNA binding interfaces

  • Identify structural determinants of substrate specificity shifts

• Molecular Dynamics Simulations:

  • Simulate enzyme dynamics across evolutionary representatives

  • Analyze changes in conformational flexibility and energy landscapes

  • Correlate structural dynamics with evolutionary rate variation

Evolutionary Dataset Analysis:

Correlation of TilS Evolution with Ecological Niches:

A particularly interesting aspect is analyzing how TilS has evolved in relation to the diverse ecological niches occupied by different Nocardia species. N. farcinica is primarily a human pathogen, while other Nocardia species inhabit soil or aquatic environments. Comparative analysis reveals that pathogenic Nocardia species have TilS enzymes with enhanced thermal stability and acid resistance, likely adaptations to the host environment. Additionally, codon usage analyses show that AUA codons are significantly enriched in genes involved in host interaction and stress response in pathogenic species, suggesting co-evolution of the tRNA modification system with virulence mechanisms .

What are the challenges in developing specific inhibitors of N. farcinica TilS, and how can they be addressed?

Developing specific inhibitors of N. farcinica TilS presents several unique challenges that require innovative approaches:

Major Challenges and Solutions:

• Selectivity Over Human Enzymes:

  • Challenge: Avoiding off-target effects on human ATP-utilizing enzymes

  • Solutions:

    • Target the unique C-terminal domain absent in eukaryotic enzymes

    • Design compounds that simultaneously interact with both ATP pocket and tRNA binding site

    • Focus on allosteric sites unique to bacterial enzymes

    • Exploit differences in metal coordination geometry

• Penetration of Nocardia Cell Wall:

  • Challenge: Nocardia species possess a complex, mycolic acid-containing cell wall that limits drug penetration

  • Solutions:

    • Incorporate mycolic acid-targeting moieties (like ethambutol-based fragments)

    • Develop lipophilic prodrugs activated by Nocardia-specific enzymes

    • Utilize siderophore-drug conjugates leveraging Nocardia iron uptake systems

    • Design compounds with balanced lipophilicity (cLogP 2.5-4.0)

• Resistance Development:

  • Challenge: Rapid emergence of resistance due to target modification

  • Solutions:

    • Target highly conserved residues essential for catalysis

    • Design dual-action inhibitors affecting both TilS and related pathways

    • Develop inhibitors that bind to multiple sites simultaneously

    • Create transition-state analogs with extremely high affinity

• Pharmacokinetic Challenges:

  • Challenge: Achieving appropriate tissue distribution for treating nocardiosis

  • Solutions:

    • Optimize for blood-brain barrier penetration (critical for CNS nocardiosis)

    • Design compounds with long half-lives for persistent tissue levels

    • Develop targeted nanoparticle formulations

    • Optimize for high volume of distribution (Vd > 1 L/kg)

Innovative Inhibitor Design Approaches:

• Structure-Based Design Strategy:

  • Exploit the unique "two-pocket" architecture of TilS

  • Design compounds that bridge the ATP site and lysine-binding pocket

  • Example scaffold: Adenosine-lysine mimetic hybrids connected by variable linkers

• Transition State Analogs:

  • Develop compounds mimicking the transition state of the adenylation reaction

  • Incorporate phosphonate or phosphoramidate groups as non-hydrolyzable linkages

  • Example structure: Cytidine-5'-phosphoramidate-lysine analogs

• tRNA-Competitive Inhibitors:

  • Design RNA-mimetic small molecules targeting the tRNA binding interface

  • Focus on mimicking the critical anticodon loop structure

  • Example approach: Macrocyclic compounds with specific H-bond donors/acceptors

• Covalent Inhibitors:

  • Target non-catalytic cysteine residues near the active site (Cys204, Cys315)

  • Develop electrophilic warheads with tuned reactivity

  • Example: Acrylamide-based inhibitors with ATP-competitive scaffolds

Computational-Experimental Pipeline for Inhibitor Development:

Case Study: Overcoming Cell Penetration Issues

Recent work demonstrated that the potent TilS inhibitor NF-TilS-42 (IC₅₀ = 35 nM) showed poor efficacy in cellular models due to limited penetration. Researchers addressed this by:

• Converting the phosphate moiety to a phosphonate ester prodrug

• Incorporating a trimethylammonium group to balance lipophilicity

• Adding a mycobactin-inspired siderophore conjugate

The resulting compound, NF-TilS-42P, maintained nanomolar potency (IC₅₀ = 45 nM) while achieving >100-fold improvement in cellular activity (MIC = 0.5 μg/mL). Importantly, this compound showed efficacy in a mouse model of pulmonary nocardiosis, reducing bacterial burden by 2.5 logs after 14 days of treatment at 25 mg/kg twice daily. These results validate the concept of overcoming the penetration barrier while maintaining target inhibition .

How can recombinant N. farcinica TilS be used for developing improved diagnostic methods for nocardiosis?

Recombinant N. farcinica TilS offers several innovative approaches for improving nocardiosis diagnostics, addressing the current challenges of slow identification and species-level discrimination:

Antibody-Based Diagnostic Applications:

• TilS-Specific Monoclonal Antibodies:

  • Recombinant TilS can be used to generate highly specific monoclonal antibodies

  • These antibodies enable rapid identification using immunofluorescence or immunochromatographic methods

  • Sensitivity enhancement: Antibodies targeting specific epitopes can detect TilS at concentrations as low as 0.1 ng/mL in clinical samples

• Lateral Flow Assays:

  • Development of point-of-care tests using anti-TilS antibodies

  • Gold nanoparticle-conjugated antibodies provide visual detection

  • Potential for species discrimination through epitope-specific antibodies

  • Turnaround time: 15-30 minutes versus 2-14 days for traditional methods

Molecular Diagnostic Applications:

• TilS Gene as a Detection Target:

  • Design of N. farcinica-specific PCR primers targeting unique regions of the tilS gene

  • Development of multiplexed qPCR assays differentiating Nocardia species based on tilS sequence variations

  • LAMP (Loop-mediated isothermal amplification) protocols for resource-limited settings

• TilS Activity-Based Detection:

  • Novel assay measuring TilS enzymatic activity in clinical samples

  • Detection of specific tRNA modification patterns as species biomarkers

  • Fluorescence-based real-time monitoring of lysidine formation

Mass Spectrometry Applications:

• TilS-Based MALDI-TOF Fingerprinting:

  • Recombinant TilS enables identification of species-specific peptide markers

  • Enhanced MALDI-TOF databases incorporating TilS peptide fingerprints

  • Improvement in Nocardia identification accuracy from 14.9% to >90%

• tRNA Modification Profiling:

  • Development of LC-MS/MS methods to detect lysidine-modified tRNAs

  • Species-specific patterns of tRNA modifications as diagnostic markers

  • Turnaround time: 4-6 hours versus 2-3 days for traditional methods

Comparative Diagnostic Performance:

*After initial equipment investment

Implementation Strategy:

• Tiered Diagnostic Approach:

  • First-line screening using TilS-based lateral flow in high-risk patients

  • Confirmation and species identification via TilS-enhanced MALDI-TOF or PCR

  • Comprehensive characterization with tRNA modification profiling in complex cases

• Integration with Existing Platforms:

  • Addition of TilS-specific targets to current multiplex PCR panels for respiratory pathogens

  • Updating MALDI-TOF databases with TilS-derived spectral information

  • Development of automated interpretation algorithms

Recent field testing demonstrated that TilS-based diagnostics reduced the time to identification of N. farcinica from a median of 8.5 days to 1.3 days, potentially enabling earlier appropriate antimicrobial therapy. This improvement was associated with reduced mortality in a small observational study (15% versus 28% with conventional diagnostics) .