Recombinant Chromobacterium violaceum Tyrosine--tRNA ligase (tyrS)

Basic Research Questions

• What is the structural classification and functional domain organization of Chromobacterium violaceum Tyrosine--tRNA ligase?

  Chromobacterium violaceum Tyrosine--tRNA ligase (tyrS), also known as Tyrosyl-tRNA synthetase (TyrRS), is classified as a class I aminoacyl-tRNA synthetase (aaRS) based on its structural and functional characteristics. The enzyme contains the defining HIGH and KMSKS consensus sequences in its active site region and features a Rossman fold catalytic domain that binds ATP and tyrosine .

  TyrRS is further sub-classified as a class Ic enzyme, functioning as an α₂ dimer unlike most class I tRNA synthetases. The protein's structure includes:

  • N-terminal catalytic domain with the Rossman fold

  • C-terminal domain involved in tRNA binding

  • Dimerization interface essential for function

  The enzyme exhibits an unusual tRNA recognition pattern, binding to tRNA^Tyr from the major groove side of the acceptor stem (similar to class II aaRSs) and binds the tRNA across both subunits in the α₂ dimer .

• What is the genomic context of the tyrS gene in Chromobacterium violaceum?

  In the Chromobacterium violaceum genome (strain ATCC 12472), the tyrS gene is designated as CV_3072 . This gene encodes the tyrosine-tRNA synthetase enzyme. The genomic context surrounding tyrS can provide insights into potential co-regulated genes or operonic structures.

  Based on KEGG database information, the tyrS gene is part of a broader network of tRNA synthetase genes in C. violaceum including:

  Gene ID	Gene Name	Function
  CV_3072	tyrS	Tyrosine-tRNA synthetase
  CV_0505	leuS	Leucyl-tRNA synthetase
  CV_3740	aspS	Aspartyl-tRNA synthetase
  CV_1962	argS	Arginyl-tRNA synthetase

  This genomic organization allows researchers to investigate potential co-regulation of aminoacyl-tRNA synthetases in response to cellular stresses or during different growth phases .

Advanced Research Questions

• What expression systems are most effective for producing recombinant C. violaceum Tyrosine--tRNA ligase?

  For the successful expression of recombinant C. violaceum Tyrosine--tRNA ligase, several expression systems have been reported with varying efficacy:

  Expression System	Advantages	Considerations
  E. coli (BL21, Rosetta)	High yield, simple cultivation	May require codon optimization
  Mammalian cells	Proper folding, post-translational modifications	Lower yield, higher cost
  Yeast	Good compromise between yield and folding	Medium complexity
  Baculovirus	High expression of complex proteins	More complex setup

  Based on available data, the optimal expression protocol involves:

  1. Transformation of expression plasmid (containing tyrS) into E. coli Rosetta 2(DE3) Singles Competent Cells

  2. Culture growth in Terrific Broth (TB) supplemented with appropriate antibiotics (50 μg/mL kanamycin, 25 μg/mL chloramphenicol)

  3. Growth at 37°C to an optical density (A600) of 0.6–0.8

  4. Induction with IPTG (500 μM)

  5. Post-induction growth for 3 hours followed by harvesting by centrifugation (10,000 g, 4°C, 45 min)

  For difficult cases, codon optimization may be necessary. In some studies with related synthetases, initial PCR amplification was unsuccessful, necessitating gene sequence optimization for expression .

• What are the key methodological approaches for determining kinetic parameters of C. violaceum Tyrosine--tRNA ligase?

  Determining accurate kinetic parameters for C. violaceum tyrS requires carefully designed experimental approaches:

  1. Aminoacylation Assay: The standard method involves measuring the rate of tRNA^Tyr aminoacylation using radioactively labeled amino acids ([³H]-tyrosine or [¹⁴C]-tyrosine). A time-course analysis yields initial velocity data for Michaelis-Menten kinetic analysis.

  2. ATP-PPi Exchange Assay: This measures the reverse reaction of amino acid activation, providing KM and kcat values for ATP and tyrosine binding independently of tRNA interaction.

  3. Scintillation Proximity Assay (SPA): This high-throughput method has been successfully used for related TyrRS enzymes and is suitable for inhibitor screening .

  For comprehensive kinetic characterization, researchers should determine:

  Parameter	Typical Range for tyrS	Experimental Approach
  KM (Tyr)	1-10 μM	Vary [Tyr] at saturating [ATP] and [tRNA]
  KM (ATP)	100-500 μM	Vary [ATP] at saturating [Tyr] and [tRNA]
  KM (tRNA^Tyr)	0.5-2 μM	Vary [tRNA] at saturating [Tyr] and [ATP]
  kcat	2-10 s⁻¹	Measure at saturating substrate concentrations
  kcat/KM	10⁶-10⁷ M⁻¹s⁻¹	Calculate from individual parameters

  The inclusion of proper controls and the use of purified components are essential for accurate measurements.

• How can structural studies of C. violaceum Tyrosine--tRNA ligase inform inhibitor design for antimicrobial development?

  Structural studies of C. violaceum tyrS can significantly advance antimicrobial development through the following approaches:

  1. X-ray Crystallography: Obtaining high-resolution crystal structures of tyrS alone and in complex with substrates (ATP, tyrosine, tRNA^Tyr) or inhibitors. This typically requires:

     • Protein concentration of >10 mg/mL

     • Screening of 500-1000 crystallization conditions

     • Co-crystallization with ligands or soaking approaches

     • Data collection at synchrotron radiation sources

  2. Structure-Based Virtual Screening: Using the ATP-binding pocket and tyrosine-binding pocket as targets for in silico screening of compound libraries.

  3. Fragment-Based Drug Design: Identifying small molecular fragments that bind to different sub-pockets within the active site.

  Studies with related TyrRS enzymes have identified several characteristics of effective inhibitors:

  Inhibitor Property	Structural Basis	Example Compounds
  ATP competitive binding	Interaction with Rossman fold	BCD38C11, BCD49D09
  Alternative mechanism	Non-substrate competitive	BCD37H06, BCD54B04
  Selective inhibition	Species-specific binding pockets	-

  The class I aminoacyl-tRNA synthetase structure of tyrS, with its conserved HIGH and KMSKS motifs, provides specific targeting opportunities. Compounds that exploit structural differences between bacterial and human TyrRS show the most promise for selective antimicrobial development .

• What role does Tyrosine--tRNA ligase play in the virulence and pathogenicity of Chromobacterium violaceum?

  While direct evidence linking tyrS to C. violaceum virulence is limited, several aspects warrant investigation:

  1. Essential Gene Function: As a crucial enzyme for protein synthesis, tyrS is essential for bacterial survival, making it a potential antibiotic target. Inhibition studies could assess its value as a therapeutic target for C. violaceum infections.

  2. Pathogenicity Context: C. violaceum causes fatal septicemia in humans and animals . The pathogenicity islands Cpi-1/-1a and Cpi-2 encode Type III secretion systems (T3SS) that are major virulence determinants , and the relationship between core metabolic genes like tyrS and virulence genes requires further study.

  3. Quorum Sensing Relationship: C. violaceum utilizes a sophisticated quorum sensing system (CviI/R) that regulates violacein production . Research could investigate whether translation-related processes involving tyrS are linked to quorum sensing responses.

  4. Biofilm Formation: C. violaceum forms biofilms that contribute to its pathogenicity . The role of protein synthesis and specifically tyrS in biofilm development represents an important research direction.

  Experimental approaches to study these relationships could include:

  • Construction of conditional tyrS mutants

  • Transcriptomic analysis of tyrS expression during infection

  • Assessment of tyrS inhibitors on virulence in animal models

  • Protein-protein interaction studies to identify non-canonical functions

• How can researchers investigate potential non-canonical functions of C. violaceum Tyrosine--tRNA ligase beyond aminoacylation?

  Aminoacyl-tRNA synthetases, including TyrRS, often exhibit functions beyond their canonical role in protein synthesis. To investigate such functions in C. violaceum tyrS:

  1. Protein Interactome Analysis:

     • Affinity purification coupled with mass spectrometry (AP-MS)

     • Bacterial two-hybrid screening

     • Co-immunoprecipitation with tyrS-specific antibodies

     • Crosslinking mass spectrometry to identify transient interactions

  2. Domain Function Analysis:

     • Generation of truncation variants to identify functional domains

     • Site-directed mutagenesis of conserved and non-conserved regions

     • Assessment of in vitro and in vivo activities beyond aminoacylation

  3. Transcriptional and Translational Regulation:

     • Chromatin immunoprecipitation to identify potential DNA-binding activities

     • RNA immunoprecipitation to identify RNA targets beyond tRNA^Tyr

     • Ribosome profiling in tyrS-depleted conditions

  4. Stress Response Studies:

     • Analysis of tyrS expression and activity under various stress conditions

     • Phenotypic analysis of tyrS overexpression or depletion under stress

  It's worth noting that in other organisms, TyrRS has been implicated in:

  Non-canonical Function	Experimental Evidence	Potential Relevance to C. violaceum
  Cytokine-like activity	Proteolytic release of cytokine domain	Potential role in host-pathogen interaction
  Transcriptional regulation	DNA binding activity	Gene regulation during stress response
  Apoptosis signaling	Nuclear translocation	Possible role in bacterial programmed cell death
  Regulatory RNA binding	RNA immunoprecipitation	Post-transcriptional regulation

  These approaches could reveal novel functions of tyrS that may contribute to C. violaceum's adaptation to environmental stresses or its virulence mechanisms.

• What are the comparative characteristics of C. violaceum Tyrosine--tRNA ligase versus other bacterial tyrS enzymes?

  Comparative analysis of C. violaceum tyrS with other bacterial orthologs provides valuable insights into evolution and potential species-specific functions:

  Species	Sequence Identity with C. violaceum tyrS	Notable Features	Inhibitor Susceptibility
  Pseudomonas aeruginosa	Varies (dual TyrRS system)	Two forms: TyrRS-Z and TyrRS-S (27% identity)	Different inhibitor profiles
  Escherichia coli	Moderate	Well-characterized structure	Susceptible to various inhibitors
  Pseudogulbenkiana ferrooxidans	High	Closely related violacein producer	Expected similar properties

  Key methodological approaches for comparative studies include:

  1. Phylogenetic Analysis:

     • Multiple sequence alignment of tyrS from diverse bacterial species

     • Construction of phylogenetic trees to establish evolutionary relationships

     • Identification of conserved versus variable regions

  2. Structural Comparison:

     • Homology modeling of C. violaceum tyrS based on crystal structures from other species

     • Superposition of structures to identify conserved binding pockets

     • Analysis of species-specific structural features that could be exploited for selective inhibition

  3. Biochemical Comparison:

     • Side-by-side kinetic analysis under identical conditions

     • Substrate specificity profiling (non-canonical amino acids, ATP analogs)

     • Temperature, pH, and salt tolerance profiles

  4. Inhibitor Cross-Reactivity:

     • Testing of known TyrRS inhibitors against multiple bacterial enzymes

     • Structure-activity relationship analysis to identify species-specific determinants

  This comparative approach is particularly relevant since some bacterial species (like P. aeruginosa) possess dual TyrRS systems, whereas C. violaceum appears to have a single tyrS gene .

• What are the most effective purification strategies for obtaining homogeneous preparations of recombinant C. violaceum Tyrosine--tRNA ligase?

  Obtaining highly pure, active preparations of recombinant C. violaceum tyrS requires a carefully designed purification strategy:

  Purification Step	Methodology	Expected Result
  Affinity Chromatography	His-tag/Ni-NTA or Strep-tag	70-80% purity
  Ion Exchange	Resource Q or S column	>85% purity
  Size Exclusion	Superdex 200	>95% purity, dimeric form
  Tag Removal	TEV or PreScission protease	Native protein

  The recommended complete purification protocol involves:

  1. Lysis Buffer Optimization:

     • 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

     • Addition of protease inhibitors (PMSF, leupeptin, pepstatin)

     • DNase I to reduce viscosity

     • Lysis via sonication or French press

  2. Affinity Purification:

     • Pre-equilibration of Ni-NTA resin

     • Batch binding followed by column packing

     • Stepwise washing with increasing imidazole (20-40 mM)

     • Elution with 250 mM imidazole

  3. Tag Cleavage and Secondary Purification:

     • Overnight dialysis with TEV protease

     • Reverse Ni-NTA to remove cleaved tag and TEV

     • Ion exchange chromatography to separate charge variants

  4. Final Polishing and Storage:

     • Size exclusion chromatography in 20 mM HEPES pH 7.5, 150 mM NaCl

     • Concentration to 1-10 mg/mL using appropriate molecular weight cutoff

     • Flash-freezing in liquid nitrogen with 10% glycerol

  Quality assessment should include SDS-PAGE (>85% purity), enzymatic activity assays, and dynamic light scattering to confirm homogeneity and proper oligomeric state .