Recombinant Chlamydophila caviae Isoleucine--tRNA ligase (ileS), partial

What is Isoleucine-tRNA ligase (ileS) and what is its essential function in bacterial metabolism?

Isoleucine-tRNA ligase (ileS), also known as isoleucyl-tRNA synthetase (IleRS), is an essential enzyme that covalently couples isoleucine to its corresponding tRNA molecules. This aminoacyl-tRNA synthetase plays a critical role in protein biosynthesis by ensuring the correct amino acid is attached to the appropriate tRNA, maintaining translational fidelity. The enzyme catalyzes a two-step reaction: first activating isoleucine with ATP to form an aminoacyl-adenylate intermediate, then transferring the activated amino acid to the 3' end of the cognate tRNA .

IleRS possesses editing mechanisms to prevent misaminoacylation of tRNA with structurally similar amino acids like valine and the non-proteinogenic norvaline, through both pre-transfer and post-transfer editing activities . These quality control mechanisms are crucial for maintaining the fidelity of protein synthesis, as even small error rates in amino acid incorporation can lead to misfolded proteins and cellular dysfunction.

How does Chlamydophila caviae ileS compare structurally and functionally to orthologs from other bacterial species?

Chlamydophila caviae ileS shares significant structural and functional similarities with other bacterial isoleucyl-tRNA synthetases while maintaining species-specific characteristics. Comparative genomic analysis reveals that C. caviae ileS is conserved among Chlamydophila species, with notable homology to Chlamydophila abortus and Chlamydophila pneumoniae orthologs .

Bacterial IleRS enzymes generally group into two distinct clades (ileS1 and ileS2) that differ in their anticodon binding domain structure, catalytic site sequence elements, and susceptibility to antibiotics like mupirocin . While most bacteria possess either ileS1 or ileS2 as a standalone housekeeping gene, some bacterial species, particularly in the Bacillaceae family, maintain both variants . The ileS2 clade typically confers resistance to mupirocin, a natural antibiotic that targets isoleucyl-tRNA synthetase .

Studies comparing the efficiency of the two IleRS variants in model organisms have demonstrated that IleRS1 can be up to 10-fold more efficient in isoleucine activation (measured as kcat/KM) compared to IleRS2, and approximately two-fold faster in Ile-tRNAIle synthesis . This functional difference may represent an evolutionary trade-off between catalytic efficiency and antibiotic resistance.

What expression systems are most effective for producing recombinant Chlamydophila caviae ileS?

Multiple expression systems have been successfully employed for the production of recombinant aminoacyl-tRNA synthetases, with selection dependent on research objectives and downstream applications. While specific data for C. caviae ileS expression is limited in the provided sources, general approaches for similar recombinant proteins can be applied.

Common expression systems include:

For functional characterization studies, E. coli-based expression systems are frequently utilized due to their efficiency and cost-effectiveness . For studies requiring native folding and post-translational modifications, eukaryotic expression systems may be preferred. Researchers should select the expression system based on the intended downstream applications and required protein characteristics.

What purification strategies yield the highest purity and activity for recombinant C. caviae ileS?

Purification of recombinant C. caviae ileS typically employs a multi-step approach to achieve high purity while maintaining enzymatic activity. Although specific protocols for C. caviae ileS are not detailed in the provided sources, standard methods for similar aminoacyl-tRNA synthetases can be recommended.

An effective purification strategy generally includes:

• Initial clarification of lysate by centrifugation and filtration

• Affinity chromatography using His-tag, GST-tag, or other affinity tags

• Ion exchange chromatography to separate based on charge properties

• Size exclusion chromatography for final polishing and buffer exchange

For functional studies, it's crucial to verify that the purified enzyme retains its aminoacylation activity. This can be assessed through aminoacylation assays measuring the enzyme's ability to charge tRNA with isoleucine. The specific activity of purified IleRS can vary significantly depending on the expression system and purification protocol employed, with E. coli-expressed enzymes often exhibiting activities comparable to those of the native enzyme when properly folded .

What are the most sensitive methodologies for assessing the catalytic activity of recombinant C. caviae ileS?

Evaluating the catalytic activity of recombinant C. caviae ileS requires assays that can quantify both steps of the aminoacylation reaction: amino acid activation and transfer to tRNA. Several methodologies with varying sensitivities and applications are available.

For amino acid activation (first step), researchers commonly employ the ATP-PPi exchange assay, which measures the formation of [32P]ATP from [32P]PPi and reflects the reversibility of the aminoacyl-adenylate formation. This method allows determination of key kinetic parameters such as kcat and KM for the substrate amino acid .

For the complete aminoacylation reaction, standard approaches include:

• Radioactive assays: Measuring the incorporation of [14C] or [3H]-labeled isoleucine into tRNA

• Thin-layer chromatography: Separating charged from uncharged tRNAs

• Filter-binding assays: Capturing aminoacyl-tRNAs on filters

• HPLC-based methods: Separating and quantifying reaction components

• Enzyme-coupled assays: Linking aminoacylation to detectable enzymatic reactions

Research has shown significant differences in catalytic efficiencies between different IleRS variants. For example, comparative studies between IleRS1 and IleRS2 in Bacilli revealed that IleRS1 is approximately 10-fold more efficient in isoleucine activation (measured as kcat/KM) and two-fold faster in the complete aminoacylation reaction . Such methodologies could be applied to investigate the catalytic properties of C. caviae ileS compared to orthologs from other species.

How does recombinant C. caviae ileS fidelity compare to orthologs from other bacterial species?

The fidelity of aminoacyl-tRNA synthetases involves their ability to discriminate between the cognate amino acid and structurally similar non-cognate amino acids. For IleRS, the primary challenge is distinguishing isoleucine from valine (which differs by a single methyl group) and the non-proteinogenic norvaline.

IleRS enzymes maintain translational fidelity through two primary editing mechanisms:

• Pre-transfer editing: Hydrolysis of non-cognate aminoacyl-adenylate intermediates within the synthetic site

• Post-transfer editing: Hydrolysis of mischarged tRNAs in a separate editing domain

Comparative studies of IleRS from different bacterial species have revealed variations in editing efficiency. While specific data for C. caviae ileS fidelity is not provided in the search results, methodological approaches for such comparisons typically include:

• Misaminoacylation assays with non-cognate amino acids (valine, norvaline)

• Pre-transfer editing assessment through analysis of AMP formation

• Post-transfer editing studies using mischarged tRNAs as substrates

• In vivo complementation studies to assess fidelity in cellular contexts

The balance between aminoacylation efficiency and editing capability often represents evolutionary adaptations to specific ecological niches or stress conditions. For example, some bacteria may sacrifice editing efficiency for antibiotic resistance, as observed in the case of ileS2 variants conferring resistance to mupirocin .

What structural and functional insights have been gained from site-directed mutagenesis studies of C. caviae ileS?

Site-directed mutagenesis has been instrumental in elucidating the structure-function relationships in aminoacyl-tRNA synthetases, including IleRS enzymes. Although the search results do not provide specific mutagenesis data for C. caviae ileS, the methodological approach and insights from related studies are relevant.

Key residues in IleRS enzymes that are typically targeted for mutagenesis include:

• Residues in the amino acid binding pocket that confer specificity for isoleucine

• Catalytic residues involved in aminoacyl-adenylate formation

• Residues in the editing domain responsible for hydrolysis of mischarged tRNAs

• tRNA recognition elements in the anticodon binding domain

Mutagenesis studies in IleRS from other species have revealed that single amino acid substitutions can dramatically alter substrate specificity, catalytic efficiency, and editing capability. For example, mutations in the editing domain can compromise fidelity, leading to increased misincorporation of non-cognate amino acids .

For C. caviae ileS, potential mutagenesis targets would include conserved residues identified through sequence alignment with well-characterized IleRS enzymes, particularly focusing on regions showing divergence between ileS1 and ileS2 clades or between Chlamydophila and other bacterial genera.

What is the significance of studying C. caviae ileS in relation to antimicrobial resistance mechanisms?

Isoleucyl-tRNA synthetase represents an important target for antimicrobial agents, and understanding the structural and functional characteristics of C. caviae ileS has significant implications for addressing antimicrobial resistance. The enzyme's essential role in protein synthesis makes it an attractive target for antibiotic development.

Mupirocin (pseudomonic acid A) is a clinically important antibiotic that specifically targets bacterial IleRS through competitive inhibition at the isoleucine binding site . Resistance to mupirocin is primarily associated with the ileS2 clade of enzymes, which show structural differences in the catalytic domain compared to the more susceptible ileS1 clade .

Comparative studies between mupirocin-sensitive and mupirocin-resistant IleRS variants can provide insights into:

• Structural determinants of antibiotic binding and resistance

• Potential for developing new IleRS inhibitors that overcome resistance

• Evolutionary relationships between different IleRS variants

• Spread and transmission of resistance genes between bacterial species

Research has demonstrated that bacteria possessing both ileS1 and ileS2 variants, such as some Bacillus species, exhibit differential expression patterns in response to stress conditions, with ileS2 being stress-induced while ileS1 is constitutively expressed . This dual system may represent an adaptive strategy balancing enzymatic efficiency with antibiotic resistance.

For Chlamydophila species, understanding the specific characteristics of ileS could inform targeted approaches for developing selective antimicrobial agents effective against these pathogens without disrupting the human microbiome.

How can molecular docking and structure-based drug design approaches be applied to C. caviae ileS for potential inhibitor development?

Molecular docking and structure-based drug design approaches provide powerful tools for identifying and optimizing potential inhibitors of C. caviae ileS. While the search results don't provide specific examples for C. caviae ileS, the methodological principles applicable to other IleRS enzymes can be extrapolated.

A comprehensive inhibitor development workflow would include:

• Structure determination or homology modeling of C. caviae ileS based on related crystal structures

• Identification of druggable pockets, including the active site and potential allosteric sites

• Virtual screening of compound libraries against identified pockets

• Medicinal chemistry optimization of initial hits

• Experimental validation through enzymatic assays and binding studies

Successful examples of this approach include the identification of small molecules that inhibit isoleucyl-tRNA synthetase from Trypanosoma brucei, which were shown to be lethal to the parasites in vitro and highly selective compared to mammalian cells . One such molecule was demonstrated to act as a competitive inhibitor of the enzyme and effectively cured mice of infection in a model system .

For C. caviae ileS, potential target sites for inhibitor design would include:

• The isoleucine binding pocket

• The ATP binding site

• The tRNA binding interface

• Editing domain active site

• Unique structural features not present in human cytoplasmic or mitochondrial IleRS

The goal would be to identify compounds with high selectivity for bacterial over human enzymes, favorable pharmacokinetic properties, and effective antimicrobial activity against Chlamydophila species.

What are the optimal conditions for assessing recombinant C. caviae ileS enzymatic activity in vitro?

Establishing optimal conditions for assessing the enzymatic activity of recombinant C. caviae ileS is crucial for accurate functional characterization. Although specific conditions for C. caviae ileS are not provided in the search results, typical parameters for IleRS activity assays can be recommended based on studies of related enzymes.

Standard reaction conditions typically include:

Time-course studies are recommended to ensure measurements are made within the linear range of the reaction. Controls should include reactions without enzyme, without ATP, or without isoleucine to account for background activity and non-specific reactions.

For comparative studies, such as those between different IleRS variants, identical conditions should be maintained to allow direct comparison of kinetic parameters. Research has shown that the cellular concentration of isoleucine (~300 μM) may allow both IleRS1 and IleRS2 to operate at near-saturating conditions in vivo .

What approaches can be used to express and purify active tRNA substrates for C. caviae ileS functional studies?

The preparation of high-quality tRNA substrates is essential for accurately characterizing the aminoacylation activity of C. caviae ileS. Several approaches are available for obtaining suitable tRNA substrates:

• Total tRNA extraction from C. caviae:

  • Phenol extraction of total RNA followed by selective precipitation of tRNA

  • Advantages: Contains native post-transcriptional modifications

  • Limitations: Low abundance of specific tRNAIle isoacceptors

• In vitro transcription of tRNAIle genes:

  • Cloning of tRNAIle genes into suitable vectors for T7 RNA polymerase transcription

  • Advantages: Homogeneous population, high yields

  • Limitations: Lacks post-transcriptional modifications that may affect recognition

• Overexpression in E. coli:

  • Co-expression of tRNA genes with modification enzymes

  • Advantages: Partial modifications, higher yields than native extraction

  • Limitations: Modification pattern may differ from the native state

For functional studies, it's essential to verify that the tRNA substrates can be effectively aminoacylated. This can be done through:

• Pilot aminoacylation assays with well-characterized IleRS enzymes

• Analysis of tRNA folding by native gel electrophoresis

• Thermal denaturation studies to assess structural stability

Research has shown that tRNA can modulate the recognition of isoleucine by IleRS enzymes, as evidenced by the differing KM values observed for isoleucine in isolated activation versus complete aminoacylation reactions . This highlights the importance of using appropriate tRNA substrates when characterizing the kinetic parameters of recombinant C. caviae ileS.

How can researchers differentiate between the editing and aminoacylation activities of recombinant C. caviae ileS?

Differentiating between the editing and aminoacylation activities of recombinant C. caviae ileS requires specialized assays targeting each function independently. These distinct activities represent crucial aspects of the enzyme's role in maintaining translational fidelity.

For assessing aminoacylation activity, standard assays include:

• Measurement of [14C]Ile-tRNAIle formation using radiolabeled isoleucine

• Quantification of AMP formation as a byproduct of aminoacylation

• Monitoring of ATP consumption through coupled enzymatic assays

For evaluating editing activity, specialized approaches include:

• Pre-transfer editing assessment:

  • Measurement of [14C]Val-AMP or [14C]Nva-AMP hydrolysis

  • Quantification of AMP formation in the presence of non-cognate amino acids

  • Analysis of ATP consumption with non-cognate versus cognate amino acids

• Post-transfer editing assessment:

  • Preparation of mischarged Val-tRNAIle or Nva-tRNAIle substrates

  • Monitoring the deacylation of mischarged tRNAs over time

  • Analysis of editing domain mutants to confirm specificity

The balance between aminoacylation and editing activities is crucial for cellular function. Research on IleRS variants has shown that there can be significant differences in both activities between different clades of the enzyme, potentially reflecting evolutionary adaptations to different selective pressures .

What are the most significant unanswered questions about C. caviae ileS that merit further investigation?

Despite advances in understanding aminoacyl-tRNA synthetases, several important questions about Chlamydophila caviae ileS remain unanswered and warrant further research:

• Structural uniqueness: How does the three-dimensional structure of C. caviae ileS differ from well-characterized bacterial IleRS enzymes, and what implications do these differences have for function and inhibitor design?

• Evolution and adaptation: Where does C. caviae ileS fall within the ileS1/ileS2 classification, and what selective pressures have shaped its evolutionary trajectory within the Chlamydophila genus?

• Substrate specificity determinants: What specific residues and structural elements determine the recognition of isoleucine versus near-cognate amino acids, and how do these compare to other bacterial IleRS enzymes?

• Role in pathogenesis: Does ileS play any specialized role in the unique developmental cycle and pathogenesis of Chlamydophila species?

• Inhibitor susceptibility: How susceptible is C. caviae ileS to known IleRS inhibitors like mupirocin, and what structural features determine this susceptibility?

• Regulation and expression: How is the expression of ileS regulated in C. caviae, particularly during different stages of its developmental cycle?

• Interaction with other cellular components: Does C. caviae ileS participate in any protein-protein interactions beyond its primary role in aminoacylation?

Addressing these questions would not only advance our understanding of this specific enzyme but could also contribute to broader knowledge about aminoacyl-tRNA synthetase evolution and function across bacterial species, potentially opening new avenues for antimicrobial development.

What are the best experimental models for studying the in vivo significance of C. caviae ileS?

Investigating the in vivo significance of C. caviae ileS presents unique challenges due to the obligate intracellular lifestyle of Chlamydophila species. Several experimental models and approaches can be considered:

• Cell culture infection models:

  • Infection of epithelial cell lines with C. caviae

  • Manipulation of ileS expression through conditional systems

  • Assessment of effects on bacterial growth and development

• Guinea pig models:

  • Natural host for C. caviae infection

  • Enables study of pathogenesis and immune response

  • Potential for testing ileS-targeted interventions

• Heterologous expression systems:

  • Expression of C. caviae ileS in E. coli with ileS knockout/knockdown

  • Assessment of complementation efficiency

  • Testing of ileS mutations on cell viability

• Comparative genomics and transcriptomics:

  • Analysis of ileS conservation across Chlamydophila species

  • Expression patterns during developmental cycle

  • Response to stress conditions and antibiotics

The methodological approaches used for studying other bacterial ileS genes, such as RNAi knockdown in model organisms or genetic complementation studies, could be adapted for investigating C. caviae ileS . Such studies would provide valuable insights into the essential nature of this enzyme for bacterial viability and its potential as a therapeutic target.