Recombinant Pseudomonas syringae pv. tomato Tyrosine--tRNA ligase (tyrS)

Basic Research Questions

• What is Pseudomonas syringae pv. tomato Tyrosine--tRNA ligase (tyrS) and what is its fundamental enzymatic function?

Pseudomonas syringae pv. tomato Tyrosine--tRNA ligase (tyrS), also known as tyrosyl-tRNA synthetase, is an essential enzyme (EC 6.1.1.1) involved in protein biosynthesis. It belongs to the aminoacyl-tRNA synthetase family and catalyzes the attachment of tyrosine to its cognate tRNA molecules through a two-step reaction:

Step 1: Tyrosine + ATP → Tyrosyl-AMP + PPi
Step 2: Tyrosyl-AMP + tRNA^Tyr → Tyrosyl-tRNA^Tyr + AMP

This reaction is critical for the accurate translation of genetic information, as it ensures that tyrosine is correctly incorporated into growing polypeptide chains during protein synthesis. The tyrS gene in P. syringae pv. tomato encodes this enzyme, which is essential for bacterial survival and pathogenicity . Similar to other bacterial tyrosyl-tRNA synthetases, the P. syringae enzyme likely functions as a homodimer or monomer with a pseudo-dimeric structure, containing a characteristic Rossmann fold typical of class I aminoacyl-tRNA synthetases .

• How does the structure of bacterial tyrosyl-tRNA synthetase relate to its function?

Bacterial tyrosyl-tRNA synthetases typically feature a modular structure with distinct functional domains:

• N-terminal Catalytic Domain (approximately 230 amino acids):

  • Contains the mononucleotide binding fold (Rossmann fold) characteristic of class I aminoacyl-tRNA synthetases

  • Houses the active site where tyrosine activation occurs

  • Contains a unique insertion between the two halves of the fold known as Connective Peptide 1 (CP1)

• C-terminal Domain:

  • Involved in tRNA recognition and binding

  • Contains anticodon recognition elements that ensure specificity

The enzyme binds tRNA^Tyr from the major groove side of the acceptor stem and binds the tRNA across both subunits in the α2 dimer. This binding occurs between the long variable arm and the anticodon stem of the tRNA, providing specificity for tRNA^Tyr and preventing the binding of non-cognate tRNAs . This recognition mechanism is crucial for maintaining translational fidelity.

The structural architecture directly supports the aminoacylation reaction, with the catalytic domain responsible for amino acid activation and the C-terminal domain ensuring correct tRNA positioning. In P. syringae pv. tomato DC3000, which has 63 tRNA genes, this specificity is particularly important for accurate protein synthesis during pathogenesis .

• What methods are used for cloning and recombinant expression of P. syringae tyrS?

While specific protocols for P. syringae pv. tomato tyrS are not extensively documented, methods established for related Pseudomonas species can be adapted. Based on successful approaches used for P. aeruginosa tyrS expression, the following methodology is recommended:

• Gene Amplification and Cloning:

  • PCR amplification of the tyrS gene from P. syringae pv. tomato genomic DNA

  • Addition of appropriate restriction sites (e.g., NheI and HindIII) to the primers

  • If direct amplification is challenging due to high GC content, codon optimization for E. coli expression may be necessary

  • Cloning into an expression vector such as pET24b(+)

• Expression Conditions:

  • Transformation into expression strains like E. coli Rosetta 2 (DE3)

  • Culture in rich media (e.g., Terrific Broth) with appropriate antibiotics

  • Induction at OD600 0.6-0.8 with IPTG (typically 0.5 mM)

  • Post-induction growth for 3-4 hours at 37°C or overnight at lower temperatures

• Protein Purification:

  • Cell lysis in buffer containing protease inhibitors

  • Initial purification by affinity chromatography (His-tag or other fusion tags)

  • Secondary purification by ion exchange chromatography

  • Final polishing by size exclusion chromatography if necessary

This approach has achieved yields of several milligrams of pure protein per liter of culture for related aminoacyl-tRNA synthetases .

• What kinetic parameters characterize bacterial tyrosyl-tRNA synthetases and how are they determined experimentally?

The kinetic properties of bacterial tyrosyl-tRNA synthetases provide insights into their catalytic efficiency and substrate specificity. Based on studies of P. aeruginosa tyrosyl-tRNA synthetases (which share structural similarities with P. syringae enzymes), the following parameters and experimental methods are relevant:

Typical Kinetic Parameters:

Methodologies for Kinetic Analysis:

• Aminoacylation Assay:

  • Reaction mixture containing varied concentrations of substrate (tyrosine, ATP, or tRNA^Tyr)

  • Constant concentrations of other components

  • Reaction initiated by addition of the limiting substrate

  • Incubation at 37°C and sampling at defined time intervals

  • Reaction terminated with EDTA (0.5 M)

• Data Analysis:

  • Initial velocities plotted against substrate concentration

  • Fitting to Michaelis-Menten equation using non-linear regression

  • Determination of Km, Vmax, and kcat

These methods allow for comprehensive characterization of the enzyme's catalytic properties and provide a foundation for structure-function studies and inhibitor development.

Intermediate Research Questions

• How does P. syringae tyrS compare with tyrosyl-tRNA synthetases from other bacterial species?

Comparative analysis of tyrosyl-tRNA synthetases across bacterial species reveals important insights into evolutionary relationships and functional adaptations:

Structural Comparison:

• Most bacterial tyrosyl-tRNA synthetases belong to Class I aminoacyl-tRNA synthetases

• They typically form homodimers or monomers with pseudo-dimeric structures

• The catalytic domain containing the Rossmann fold is highly conserved

• Greater variability exists in the C-terminal domain involved in tRNA recognition

Functional Differences:

• Some species (like B. subtilis) possess two forms of tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z)

• P. aeruginosa TyrRS-S and TyrRS-Z show different kinetic parameters:

  • TyrRS-S: Km values of 172 μM (Tyr), 204 μM (ATP), and 1.5 μM (tRNA^Tyr)

  • TyrRS-Z: Km values of 29 μM (Tyr), 496 μM (ATP), and 1.9 μM (tRNA^Tyr)

The presence of multiple TyrRS forms in some Pseudomonas species suggests potential specialized functions. In B. subtilis, TyrRS-Z has increased selectivity for L-tyrosine over D-tyrosine, preventing misincorporation during stationary phase . Whether P. syringae has similar adaptations remains to be fully investigated, but could be relevant to its lifecycle as a plant pathogen with varying environmental conditions.

• What role might tyrS play in P. syringae pathogenicity and plant-microbe interactions?

While the direct role of tyrS in P. syringae pathogenicity has not been extensively characterized, several lines of evidence suggest potential contributions to virulence:

• Essential Role in Protein Synthesis:

  • As a crucial enzyme for translation, tyrS is essential for the synthesis of virulence factors

  • Proper protein synthesis is required for the expression of pathogenicity determinants such as type III secretion system components and phytotoxins

• Potential Adaptation to Host Environment:

  • The plant environment presents specific challenges including altered nutrient availability and defense responses

  • P. syringae pv. tomato DC3000 delivers approximately 30 type III effector proteins into host cells to suppress plant immunity

  • Efficient translation is required for production of these virulence factors

• Stress Response and Adaptation:

  • Studies in P. syringae show that protein tyrosine kinase activity varies with temperature and growth phase

  • Expression is higher at elevated temperatures (22°C vs. 4°C) and during stationary phase

  • Similar regulatory mechanisms might apply to tyrS function in response to environmental changes

• Indirect Effects on Virulence:

  • Efficient translation is crucial for bacterial population growth in planta

  • Rapid adaptation to changing conditions during infection requires robust protein synthesis machinery

  • Coronatine, a major P. syringae phytotoxin, requires numerous enzymes whose synthesis depends on functional translation machinery

Understanding the role of tyrS in pathogenicity could provide insights into basic aspects of P. syringae biology and potentially reveal new approaches for disease management.

• What challenges exist in purifying active recombinant P. syringae tyrS, and how can they be addressed?

Purification of active recombinant P. syringae tyrS presents several technical challenges that require specific strategies:

• Solubility and Folding Issues:
  Challenge: Overexpressed bacterial proteins often form inclusion bodies
  Solutions:

  • Optimize expression temperature (16-25°C rather than 37°C)

  • Use solubility-enhancing fusion tags

  • Co-express with chaperones

  • Add solubility enhancers to culture media

• Enzyme Activity Preservation:
  Challenge: Maintaining enzymatic activity throughout purification
  Solutions:

  • Include reducing agents (DTT) in all buffers

  • Maintain constant cold temperature during purification (4°C)

  • Add glycerol (10-20%) to stabilize protein structure

  • Include protease inhibitors to prevent degradation

  • Minimize freeze-thaw cycles by aliquoting purified protein

• Contaminating Activities:
  Challenge: Removing host E. coli aminoacyl-tRNA synthetases
  Solutions:

  • Design affinity tags that allow highly specific purification

  • Implement multiple chromatography steps

  • Use species-specific tRNA^Tyr for activity assays

For recombinant tyrosyl-tRNA ligase expression, buffer composition is critical. A typical stabilizing buffer might contain:

• 25 mM Tris-HCl (pH 7.3)

• 100 mM glycine

• 10% glycerol

By implementing these strategies, researchers can typically achieve yields of pure, active enzyme suitable for biochemical and structural studies. For human tyrosinase (which uses similar purification principles), yields of 4-6 mg of pure protein per liter of culture have been reported .

• How can substrate specificity of P. syringae tyrS be assessed experimentally?

Assessing the substrate specificity of P. syringae tyrS requires methodical experimental approaches that examine its interaction with various substrates and potential analogs:

• Amino Acid Specificity:
  Methodology:

  • ATP-PPi exchange assay with various amino acids

  • Aminoacylation assays with radiolabeled amino acids

  • Competition assays with tyrosine and structurally related amino acids

• tRNA Recognition:
  Methodology:

  • In vitro transcription to generate tRNA^Tyr and mutant variants

  • Aminoacylation assays with various tRNA species

  • Binding assays using gel shift or surface plasmon resonance

• ATP/GTP Utilization:
  Methodology:

  • Aminoacylation assays with ATP, GTP, or other nucleotides

  • Kinetic analysis with varying nucleotide concentrations

  Testing for ATP/GTP specificity is particularly important as kinetic parameters for P. aeruginosa TyrRS forms show significant differences in their interaction with ATP:

  • TyrRS-S: Km(ATP) = 204 μM, kcat = 1.0 s^-1

  • TyrRS-Z: Km(ATP) = 496 μM, kcat = 3.8 s^-1

• Inhibitor Studies:

  Inhibition studies can provide valuable insights into substrate binding mechanisms. Studies with P. aeruginosa TyrRS identified compounds that inhibit by different mechanisms:

  • BCD38C11 and BCD49D09: ATP-competitive inhibitors

  • BCD37H06 and BCD54B04: Non-substrate competitive inhibitors

  Similar approaches could be applied to P. syringae tyrS to characterize its substrate binding sites.

These experimental approaches provide comprehensive insights into substrate specificity determinants of P. syringae tyrS, with implications for understanding its evolutionary adaptation and potential vulnerability to inhibitors.

Advanced Research Questions

• What potential exists for developing specific inhibitors of P. syringae tyrS, and how might this approach contribute to plant disease management?

Developing specific inhibitors of P. syringae tyrS represents a promising approach for targeted antimicrobial development with applications in plant disease management:

• Rationale for tyrS as a Target:

  • Essential enzyme for bacterial protein synthesis

  • Structural differences between bacterial and eukaryotic tyrosyl-tRNA synthetases

  • Potential to develop compounds with specificity for bacterial enzymes

• Inhibitor Discovery Strategies:

  • High-throughput screening using enzymatic assays

    • Scintillation proximity assay (SPA) technology has been successful for P. aeruginosa TyrRS

    • Similar screening approaches could be applied to P. syringae tyrS

  • Structure-based design leveraging crystal structures

  • Fragment-based screening

• Classes of Potential Inhibitors:

  • ATP-competitive inhibitors

    • In P. aeruginosa studies, compounds BCD38C11 and BCD49D09 were identified as ATP-competitive inhibitors

  • Tyrosine-competitive analogs

  • Allosteric inhibitors affecting enzyme dynamics

• Efficacy Data from Related Systems:

  Compound	Target	IC50 Value	Inhibition Mechanism	Antimicrobial Activity
  BCD37H06	P. aeruginosa TyrRS-S	24 μM	Non-substrate competitive	Broad-spectrum
  BCD38C11	P. aeruginosa TyrRS-S	71 μM	ATP-competitive	Broad-spectrum
  BCD49D09	P. aeruginosa TyrRS-S	65 μM	ATP-competitive	Broad-spectrum
  BCD54B04	P. aeruginosa TyrRS-S	50 μM	Non-substrate competitive	Broad-spectrum
  BCD38C11	P. aeruginosa TyrRS-Z	241 μM	ATP-competitive	Broad-spectrum

• Potential Applications in Plant Disease Management:

  • Preventative applications before infection

  • Treatment of established infections

  • Seed treatments to protect during germination

  • Integration with existing disease management strategies

Research on P. aeruginosa tyrosyl-tRNA synthetase inhibitors has identified compounds with IC50 values as low as 24 μM that show broad-spectrum activity against multiple bacterial pathogens without cytotoxicity to human cells at concentrations up to 400 μg/mL . Similar approaches could yield effective inhibitors for P. syringae tyrS.

• How can structural biology approaches advance our understanding of P. syringae tyrS function and inhibition?

Structural biology approaches offer powerful tools for elucidating the molecular mechanisms of P. syringae tyrS function and for rational inhibitor design:

These approaches have been successfully applied to tyrosyl-tRNA synthetases from other organisms. For example, human tyrosinase expression and purification methods have been optimized to obtain high yields of pure protein required for crystallization trials . Similar methodologies could be adapted for structural studies of P. syringae tyrS.

• What is the role of tyrS in bacterial stress response and adaptation, and how can this be studied experimentally?

Aminoacyl-tRNA synthetases, including tyrS, play complex roles in bacterial stress response beyond their canonical function in protein synthesis:

• Expression Regulation Under Stress Conditions:
  Evidence:

  • Studies in P. syringae show differential expression of translation machinery components under stress

  • Protein tyrosine kinase activity in P. syringae varies with temperature and growth phase

  Experimental Approaches:

  • qRT-PCR to measure tyrS transcript levels under various stresses

  • Western blotting to quantify protein levels

  • Reporter gene fusions (e.g., tyrS promoter-GFP) to monitor expression in real-time

  • RNA-seq to place tyrS regulation in the context of global transcriptional responses

• Post-Translational Regulation:
  Evidence:

  • P. syringae contains protein kinases capable of tyrosine phosphorylation

  • The Antarctic psychrotrophic bacterium P. syringae contains a 66-kDa cytoplasmic protein phosphorylated on tyrosine residues

  Experimental Approaches:

  • Phosphoproteomic analysis under different growth conditions

  • In vitro phosphorylation assays

  • Site-directed mutagenesis of potential modification sites

  • Activity assays comparing native and modified forms of the enzyme

• Contribution to Stress Tolerance:
  Evidence:

  • Translation quality control is crucial during stress exposure

  • P. syringae protein tyrosine kinase showed higher expression at 22°C than at 4°C, and during stationary phase growth

  Experimental Approaches:

  • Construction of conditional mutants with altered tyrS expression

  • Assessment of growth and survival under various stresses

  • Competition assays between wild-type and tyrS-altered strains

  • Proteomic analysis to detect changes in mistranslation rates

These experimental approaches can reveal how tyrS contributes to P. syringae adaptation to different environments, including the plant host during pathogenesis and under various environmental stresses.

• How can advanced mutagenesis approaches be used to investigate structure-function relationships in P. syringae tyrS?

Advanced mutagenesis approaches provide powerful tools for dissecting the structural determinants of tyrS function, substrate specificity, and potential for inhibitor development:

• Site-Directed Mutagenesis for Active Site Analysis:
  Methodology:

  • Identification of conserved residues from sequence alignments and structural data

  • PCR-based site-directed mutagenesis

  • Expression and purification of mutant proteins

  • Comprehensive kinetic characterization

  Key Targets:

  • Catalytic residues (HIGH and KMSKS motifs)

  • Tyrosine binding pocket residues

  • ATP binding site

  • tRNA contact points

• Alanine-Scanning Mutagenesis:
  Methodology:

  • Systematic replacement of surface residues with alanine

  • High-throughput expression and activity screening

  • Detailed analysis of functionally important regions

  Applications:

  • Mapping of protein-protein interaction sites

  • Identification of allosteric sites

  • Discovery of unexpected functional regions

• Domain Swapping and Chimeric Proteins:
  Methodology:

  • Construction of chimeras with domains from other bacterial tyrosyl-tRNA synthetases

  • Expression and functional characterization

  • Structural analysis of chimeric proteins

  Applications:

  • Delineation of domain functions

  • Investigation of species-specific properties

  • Engineering enzymes with desired characteristics

• Experimental Predictions for Structure-Function Analysis:

  Mutation Target	Predicted Function	Effect on Tyrosine Binding	Effect on ATP Binding	Effect on tRNA Binding	Effect on kcat
  HIGH motif histidine	ATP binding	Minimal	Severe decrease	Minimal	Severe decrease
  KMSKS motif lysines	ATP binding, transition state stabilization	Minimal	Moderate decrease	Moderate decrease	Severe decrease
  Tyrosine binding pocket residues	Amino acid recognition	Severe decrease	Minimal	Minimal	Moderate decrease
  CP1 domain residues	tRNA positioning	Minimal	Minimal	Moderate decrease	Variable effects
  Dimer interface residues	Quaternary structure stabilization	Variable effects	Variable effects	Moderate decrease	Variable effects

These mutagenesis approaches, when combined with structural and functional analyses, provide comprehensive insights into the molecular basis of tyrS function and evolution, with implications for inhibitor design and protein engineering applications targeting P. syringae as a plant pathogen.