Recombinant Protochlamydia amoebophila Isoleucine--tRNA ligase (ileS), partial

What is isoleucine-tRNA ligase and what function does it serve in Protochlamydia amoebophila?

Isoleucine-tRNA ligase (ileS), also known as isoleucyl-tRNA synthetase (IleRS), is an essential enzyme responsible for catalyzing the attachment of isoleucine to its cognate tRNA molecules during protein synthesis. In Protochlamydia amoebophila, as in other organisms, this enzyme plays a critical role in the accurate translation of genetic information by ensuring that isoleucine is correctly incorporated into growing polypeptide chains. The enzyme specifically catalyzes the ATP-dependent esterification of isoleucine to the 3′-terminal adenosine of tRNA^Ile . This reaction is crucial for maintaining translational fidelity and represents one of the key steps in the process of tRNA charging that precedes protein synthesis on the ribosome.

How does the isoleucine-tRNA ligase from Protochlamydia amoebophila compare to that of other bacterial species?

The isoleucine-tRNA ligase from Protochlamydia amoebophila exhibits structural and functional similarities to other bacterial IleRS enzymes but also possesses distinctive characteristics. Unlike the IleRS found in Chlamydiaceae family members that infect mammalian hosts, the P. amoebophila enzyme does not show evidence of up-selection for tryptophan residues . This is particularly noteworthy as P. amoebophila resides in protozoan rather than mammalian hosts, suggesting evolutionary adaptation to different environmental constraints. When comparing protein-to-proteome tryptophan ratios (p/P Trp ratio), P. amoebophila does not demonstrate the tryptophan enrichment pattern observed in mammalian-infecting chlamydial species, indicating different selective pressures acting on the enzyme's amino acid composition .

What is the significance of tRNA charging in bacterial survival and persistence?

tRNA charging, facilitated by aminoacyl-tRNA synthetases like isoleucine-tRNA ligase, is fundamental to bacterial protein synthesis and therefore essential for survival. The process ensures the correct pairing of amino acids with their corresponding tRNAs, maintaining translational accuracy. In organisms like Protochlamydia amoebophila, efficient tRNA charging is particularly important during various growth phases and stress conditions. For Chlamydiaceae family members, the efficiency of tRNA charging, especially under amino acid limitation conditions, can influence their ability to enter and maintain a persistent state . The regulation of aminoacyl-tRNA synthetase expression and activity represents a critical control point for bacterial adaptation to environmental challenges, including nutrient limitation or host immune responses.

What catalytic reactions are performed by isoleucine-tRNA ligase?

Isoleucine-tRNA ligase catalyzes several reactions that ensure the accurate attachment of isoleucine to tRNA^Ile. Based on what we know about this enzyme family from other organisms like E. coli, the primary reaction is:

tRNA^Ile + L-isoleucine + ATP → L-isoleucyl-[tRNA^Ile] + AMP + diphosphate

Additionally, the enzyme possesses editing capabilities to correct misacylation events. When valine is erroneously attached to tRNA^Ile, the enzyme can catalyze hydrolysis of the incorrect aminoacyl-tRNA:

L-valyl-[tRNA^Ile] + H₂O → tRNA^Ile + L-valine + H⁺

This proofreading function is crucial for maintaining translational fidelity, as the incorporation of incorrect amino acids into proteins could lead to misfolding and dysfunction.

How does tryptophan content influence the expression and function of isoleucine-tRNA ligase in Protochlamydia amoebophila compared to other Chlamydial species?

The tryptophan content of isoleucine-tRNA ligase in Protochlamydia amoebophila represents an important factor in understanding its expression patterns, particularly when compared to mammalian-infecting chlamydial species. Unlike the Chlamydiaceae family members that exhibit up-Trp selection (elevated tryptophan content relative to their proteome average), P. amoebophila does not demonstrate this characteristic . This difference has significant implications for enzyme expression under tryptophan-limited conditions.

The lack of up-Trp selection in P. amoebophila's isoleucine-tRNA ligase suggests that this enzyme is less susceptible to translational inhibition during tryptophan starvation compared to its counterparts in mammalian-infecting chlamydial species. This evolutionary distinction likely reflects adaptation to different host environments, as P. amoebophila resides in protozoan hosts where tryptophan availability may be more consistent than in mammalian hosts where interferon-γ-induced indoleamine 2,3-dioxygenase can deplete tryptophan as a host defense mechanism .

What structural domains characterize the isoleucine-tRNA ligase in Protochlamydia amoebophila, and how do they contribute to substrate specificity?

Isoleucine-tRNA ligase in P. amoebophila, like other class I aminoacyl-tRNA synthetases, is expected to contain several conserved structural domains that collectively contribute to its substrate specificity and catalytic function. While specific structural data for the P. amoebophila enzyme is limited in the search results, comparative analysis with other bacterial isoleucine-tRNA ligases suggests the presence of:

• A Rossmann fold catalytic domain containing the HIGH and KMSKS motifs characteristic of class I aminoacyl-tRNA synthetases

• A connective polypeptide (CP) domain involved in tRNA binding

• An editing domain responsible for hydrolyzing misacylated tRNA species

• A C-terminal domain that provides additional tRNA interaction surfaces

These domains work in concert to ensure accurate discrimination between isoleucine and structurally similar amino acids like valine and leucine, thereby maintaining translational fidelity. The specific arrangement and sequence composition of these domains in P. amoebophila would reflect evolutionary adaptations to its unique ecological niche and host environment.

How can researchers interpret the impact of amino acid starvation on isoleucine-tRNA ligase activity in Protochlamydia amoebophila?

The impact of amino acid starvation, particularly tryptophan limitation, on isoleucine-tRNA ligase activity in P. amoebophila should be interpreted in the context of its evolutionary history and protein composition. Unlike mammalian-infecting chlamydial species that show pronounced sensitivity to tryptophan depletion, P. amoebophila appears to have evolved without the same degree of tryptophan dependency in its isoleucine-tRNA ligase .

Researchers should consider several key factors when interpreting amino acid starvation effects:

• Proteomic tryptophan content: P. amoebophila's isoleucine-tRNA ligase lacks the elevated tryptophan content seen in mammalian-infecting chlamydial species, suggesting potentially different regulatory mechanisms.

• Growth kinetics: Changes in growth rate during amino acid limitation should be correlated with measurements of tRNA charging efficiency to establish causative relationships.

• Comparative analysis: Experiments should include multiple chlamydial species to highlight the unique response patterns of P. amoebophila.

• Energy metabolism connections: Since aminoacyl-tRNA synthetase activity is ATP-dependent, amino acid starvation effects may be compounded by energy metabolism disruptions.

These interpretative frameworks allow researchers to distinguish direct effects on isoleucine-tRNA ligase from broader metabolic perturbations during amino acid limitation experiments.

What is the relationship between GTP availability and isoleucine-tRNA ligase function in bacterial translation systems?

While isoleucine-tRNA ligase itself utilizes ATP rather than GTP for its aminoacylation reaction, GTP availability indirectly affects the functional context in which this enzyme operates. GTP serves as an energy source during the elongation stage of translation, being required for:

• The binding of aminoacyl-tRNA to the ribosomal A site

• The translocation of the ribosome along the mRNA template

In Chlamydiae, including P. amoebophila, GTP import is facilitated by broad-specificity translocases, highlighting the importance of this nucleotide for translational processes . Additionally, GTP in chlamydial species serves as a substrate for GTP-dependent phosphoenolpyruvate carboxykinase, potentially linking translation efficiency to central carbon metabolism .

Therefore, while isoleucine-tRNA ligase doesn't directly utilize GTP, its functional output (charged tRNA^Ile) depends on GTP availability for subsequent steps in the translation process. This relationship becomes particularly important under stress conditions where energy metabolism may be compromised.

What expression systems are most effective for producing recombinant Protochlamydia amoebophila isoleucine-tRNA ligase?

For recombinant expression of P. amoebophila isoleucine-tRNA ligase, several expression systems can be considered, each with specific advantages:

• E. coli expression systems: The most widely used approach employs E. coli BL21(DE3) or similar strains with T7 RNA polymerase-based expression vectors. For optimal expression, codon optimization may be necessary to account for the different codon usage bias between P. amoebophila and E. coli. Expression can be enhanced using specialized strains that supply rare tRNAs (e.g., Rosetta or CodonPlus strains).

• Cell-free expression systems: These provide advantages for potentially toxic proteins and allow direct incorporation of modified amino acids. A purified E. coli-based cell-free system could be particularly useful for mechanistic studies requiring specifically labeled enzyme variants.

• Insect cell expression: For cases where E. coli-produced enzyme lacks proper folding or activity, baculovirus-mediated expression in insect cells (Sf9 or Hi5) represents an alternative that often yields higher amounts of properly folded protein.

The choice of expression system should be guided by the intended application, required protein purity, and whether post-translational modifications are expected to influence enzyme function.

What purification strategies yield the highest activity for recombinant isoleucine-tRNA ligase?

Purification of recombinant P. amoebophila isoleucine-tRNA ligase requires a multi-step approach to maintain enzyme activity while achieving high purity:

• Affinity chromatography: Expression with an N-terminal or C-terminal polyhistidine tag allows initial purification using immobilized metal affinity chromatography (IMAC). Buffer conditions should include 5-10% glycerol and reducing agents (DTT or β-mercaptoethanol) to maintain protein stability.

• Ion exchange chromatography: A second purification step using ion exchange (typically Q-Sepharose) can separate the target protein from contaminants with different charge properties.

• Size exclusion chromatography: A final polishing step using gel filtration separates aggregates and provides buffer exchange into the storage buffer.

Throughout purification, it's crucial to:

• Maintain temperatures between 4-10°C

• Include protease inhibitors in early purification steps

• Test activity after each purification stage to identify steps that may compromise enzyme function

• Consider including ATP in buffers at low concentrations (0.1-0.5 mM) to stabilize the enzyme

The purified enzyme should be stored with 20-30% glycerol at -80°C in small aliquots to maintain activity through multiple freeze-thaw cycles.

How can researchers accurately measure the aminoacylation activity of isoleucine-tRNA ligase?

Several complementary methods can be used to accurately measure the aminoacylation activity of isoleucine-tRNA ligase:

• Radioactive assay: The traditional gold standard involves measuring the incorporation of radioactively labeled isoleucine ([³H] or [¹⁴C]-isoleucine) into tRNA. After the reaction, tRNA is precipitated with trichloroacetic acid, collected on filters, and the radioactivity is measured by scintillation counting.

• Pyrophosphate release assay: This continuous spectrophotometric assay couples the release of pyrophosphate during aminoacylation to the oxidation of NADH through a series of enzymatic reactions, allowing real-time monitoring of activity.

• ATP consumption assay: The consumption of ATP can be monitored using a coupled enzyme system with pyruvate kinase and lactate dehydrogenase, measuring the decrease in NADH absorbance at 340 nm.

• Mass spectrometry: High-resolution mass spectrometry can directly detect the mass shift associated with tRNA aminoacylation, providing a label-free method for assessing enzyme activity.

For kinetic analysis, reactions should be performed under conditions where:

• Substrate concentrations are varied systematically

• Initial velocity conditions are maintained (<10% substrate consumption)

• Temperature and pH are carefully controlled

• Enzyme concentrations are in the linear response range

Standard reaction conditions typically include:

• 50-100 mM HEPES or Tris buffer (pH 7.5-8.0)

• 10-20 mM MgCl₂

• 1-5 mM ATP

• 50-200 μM isoleucine

• 1-5 μM tRNA^Ile

• 0.1-1 μM enzyme

What approaches can be used to study the incorporation of modified nucleosides in the context of isoleucine-tRNA ligase research?

Studying modified nucleosides in the context of isoleucine-tRNA ligase research involves several specialized approaches:

• In vitro transcription with modified NTPs: Modified nucleoside triphosphates can be incorporated into tRNA transcripts using T7 RNA polymerase, allowing the production of tRNAs with site-specific modifications for aminoacylation studies .

• Enzymatic post-transcriptional modification: tRNA modification enzymes can be used to introduce specific modifications into in vitro transcribed tRNAs, creating substrates that more closely resemble native tRNAs.

• Primer extension assays: DNA polymerases such as KOD, Thermococcus sp. 91N, and KTq have been shown to incorporate modified nucleotides, allowing the study of DNA-protein interactions that might be relevant to understanding RNA-protein interactions .

• Mass spectrometry analysis: To characterize the incorporation of modified nucleosides, liquid chromatography-mass spectrometry (LC-MS) provides definitive identification and quantification of modified nucleosides in tRNA molecules.

• Fluorescence-based approaches: Fluorescently labeled nucleotides can be incorporated into tRNAs to study binding dynamics with isoleucine-tRNA ligase through fluorescence anisotropy or FRET-based assays.

These techniques allow researchers to investigate how tRNA modifications affect recognition and aminoacylation by isoleucine-tRNA ligase, providing insights into the molecular basis of enzyme specificity.

How should researchers interpret differences in tryptophan content between isoleucine-tRNA ligase and other proteins in Protochlamydia amoebophila?

When interpreting differences in tryptophan content between isoleucine-tRNA ligase and other proteins in P. amoebophila, researchers should consider several quantitative metrics and comparative frameworks:

• Protein/Proteome (p/P) Trp ratio analysis: This metric normalizes the tryptophan content of a specific protein to the average tryptophan content of the entire proteome. For accurate interpretation, researchers should calculate:

  • The raw percentage of tryptophan residues in the protein

  • The average tryptophan percentage across the entire proteome

  • The ratio between these values (p/P Trp ratio)

A p/P Trp ratio near 1.0 suggests no selection pressure for altered tryptophan content, while values significantly above or below 1.0 indicate up-Trp or down-Trp selection, respectively.

• Cross-species comparison: The p/P Trp ratio of isoleucine-tRNA ligase should be compared across related chlamydial species to identify evolutionary patterns. Unlike mammalian-infecting Chlamydiaceae that show up-Trp selection for certain proteins, P. amoebophila generally does not exhibit this pattern .

• Functional context interpretation: Tryptophan distribution patterns should be interpreted in the context of protein function. In P. amoebophila, the lack of up-Trp selection in isoleucine-tRNA ligase suggests this enzyme would maintain expression levels during tryptophan limitation, potentially conferring a selective advantage in its natural ecological niche.

The table below illustrates how to organize and interpret tryptophan content data across different proteins and organisms:

What kinetic parameters are most relevant when characterizing recombinant isoleucine-tRNA ligase?

The characterization of recombinant isoleucine-tRNA ligase should include determination of several key kinetic parameters:

• Substrate-specific parameters:

  • K₍ₘ₎ for isoleucine: Typically in the low micromolar range (5-50 μM)

  • K₍ₘ₎ for ATP: Usually in the 100-500 μM range

  • K₍ₘ₎ for tRNA^Ile: Often in the 0.5-5 μM range

  • k₍cat₎: Turnover number, typically 1-10 s⁻¹

  • k₍cat₎/K₍ₘ₎: Catalytic efficiency, important for comparing wild-type and mutant enzymes

• Editing parameters:

  • K₍ₘ₎ for misacylated tRNA (e.g., Val-tRNA^Ile)

  • k₍cat₎ for hydrolysis of misacylated tRNA

  • Misacylation frequency with non-cognate amino acids

• Inhibition constants:

  • K₍i₎ for competitive inhibitors

  • IC₅₀ values for various inhibitors

For comprehensive characterization, these parameters should be determined under various conditions:

• Different pH values (typically pH 6.5-8.5)

• Various temperatures (ideally including the physiological temperature of the host organism)

• Different ionic strengths

• In the presence of potential regulators or inhibitors

The data should be fitted to appropriate enzyme kinetic models (Michaelis-Menten, substrate inhibition, etc.) using non-linear regression, and statistical analysis should include calculation of standard errors and confidence intervals for all parameters.

How does amino acid composition analysis inform our understanding of evolutionary adaptations in Protochlamydia amoebophila proteins?

Amino acid composition analysis, particularly focusing on tryptophan content, provides valuable insights into the evolutionary adaptations of P. amoebophila proteins:

These analytical approaches allow researchers to move beyond sequence homology to understand the selective forces that shaped P. amoebophila's proteome during its evolutionary history.

What experimental controls are essential when studying recombinant Protochlamydia amoebophila isoleucine-tRNA ligase?

Rigorous experimental design for recombinant P. amoebophila isoleucine-tRNA ligase research requires several essential controls:

• Enzyme activity controls:

  • Positive control: Commercial or well-characterized E. coli isoleucine-tRNA ligase

  • Negative control: Heat-inactivated enzyme preparation

  • Buffer control: Reaction mixture lacking the enzyme

  • Substrate controls: Reactions lacking individual substrates (ATP, isoleucine, or tRNA)

• Specificity controls:

  • Cross-aminoacylation test: Assessing activity with non-cognate amino acids (valine, leucine)

  • tRNA specificity test: Measuring activity with non-cognate tRNAs

• Expression and purification controls:

  • Empty vector control: Host cells transformed with expression vector lacking the insert

  • Wild-type reference: Comparison to wild-type enzyme when studying mutant variants

  • Endotoxin testing: Ensuring preparations are free from host cell contaminants that could affect assays

• Stability and storage controls:

  • Time-course stability assay: Testing enzyme activity after various storage periods

  • Fresh vs. frozen comparison: Establishing the impact of freeze-thaw cycles

  • Buffer composition effects: Testing activity in different storage buffers

• Analytical controls:

  • Standard curves: For all quantitative measurements

  • Internal standards: Especially for mass spectrometry-based analyses

  • Technical and biological replicates: Minimum of three for statistical validity

Implementation of these controls ensures that experimental observations can be reliably attributed to the properties of the recombinant enzyme rather than artifacts or experimental variables.

How does the tryptophan content of isoleucine-tRNA ligase compare across different bacterial species, and what does this reveal about evolutionary adaptation?

Comparative analysis of isoleucine-tRNA ligase tryptophan content across bacterial species reveals distinct patterns of evolutionary adaptation:

The tryptophan content of isoleucine-tRNA ligase varies significantly between different bacterial lineages, reflecting adaptation to diverse ecological niches. This variation is particularly evident when comparing P. amoebophila with members of the Chlamydiaceae family:

This pattern suggests that:

• Mammalian-infecting chlamydial species show moderate up-Trp selection in their isoleucine-tRNA ligase, potentially as part of a broader adaptation to environmental conditions where tryptophan limitation serves as a regulatory cue .

• P. amoebophila, which infects protozoan hosts, shows no significant selection for altered tryptophan content, suggesting different evolutionary pressures in this host environment .

• Free-living bacteria like E. coli typically exhibit down-Trp selection for isoleucine-tRNA ligase relative to their proteome average, possibly reflecting selection for translational efficiency in proteins essential for basic cellular functions .

These differences correlate with the host environment and lifestyle of each organism, highlighting how selective pressures related to amino acid availability have shaped the evolution of aminoacyl-tRNA synthetases across bacterial lineages.

What insights can structural bioinformatics provide about the functional domains of Protochlamydia amoebophila isoleucine-tRNA ligase?

Structural bioinformatics approaches can provide valuable insights into the functional domains of P. amoebophila isoleucine-tRNA ligase, even in the absence of a crystal structure:

• Domain architecture prediction: Sequence-based analysis would likely reveal the typical class I aminoacyl-tRNA synthetase architecture, including:

  • N-terminal catalytic domain with Rossmann fold

  • Connective peptide (CP) domain

  • Editing domain

  • C-terminal anticodon-binding domain

• Catalytic site conservation: Multiple sequence alignment with structurally characterized isoleucine-tRNA ligases would reveal conservation of critical catalytic residues, including:

  • The HIGH motif involved in ATP binding

  • The KMSKS motif that participates in transition state stabilization

  • Residues that form the isoleucine-binding pocket

• Editing domain analysis: Comparative modeling could identify the conserved threonine residue in the editing domain that is critical for hydrolyzing misacylated Val-tRNA^Ile, helping to understand the enzyme's fidelity mechanisms.

• Electrostatic surface mapping: Homology modeling combined with electrostatic potential calculation would reveal positively charged surfaces likely involved in tRNA binding.

• Molecular dynamics simulations: These could provide insights into conformational changes associated with substrate binding and catalysis, particularly the relative movements of domains during the aminoacylation reaction.

• Conservation mapping: Mapping sequence conservation onto structural models would identify regions under strong evolutionary constraint, highlighting functionally important surfaces beyond the active site.

These complementary bioinformatic approaches would enable researchers to generate testable hypotheses about structure-function relationships in P. amoebophila isoleucine-tRNA ligase, guiding experimental design for mutagenesis and functional studies.

How can researchers effectively compare the kinetic properties of wild-type and mutant variants of isoleucine-tRNA ligase?

Effective comparison of wild-type and mutant isoleucine-tRNA ligase variants requires a comprehensive kinetic characterization approach:

For meaningful interpretation, mutation effects should be categorized as affecting:

• Substrate binding (changes in K₍ₘ₎)

• Catalytic efficiency (changes in k₍cat₎)

• Conformational dynamics (changes in thermostability or cooperativity)

• Editing function (changes in misacylation frequency)

This systematic approach enables researchers to pinpoint the specific mechanistic consequences of mutations, providing insights into structure-function relationships within the enzyme.

What are the most promising future research directions for studying Protochlamydia amoebophila isoleucine-tRNA ligase?

Future research on P. amoebophila isoleucine-tRNA ligase holds promise in several key directions:

• Structural biology: Determination of the three-dimensional structure of P. amoebophila isoleucine-tRNA ligase through X-ray crystallography or cryo-electron microscopy would provide invaluable insights into its unique features compared to other bacterial orthologs.

• Tryptophan content engineering: Creating variant enzymes with altered tryptophan content could test hypotheses about the relationship between amino acid composition and enzyme function/regulation under limitation conditions. This experimental approach could validate computational predictions about the significance of p/P Trp ratios .

• Host-specific adaptation studies: Comparative analysis of isoleucine-tRNA ligase function in different host environments could reveal how this essential enzyme adapts to diverse ecological niches, particularly comparing P. amoebophila's protozoan host environment with the mammalian host environment of Chlamydiaceae species .

• Integration with systems biology: Exploring the role of isoleucine-tRNA ligase in the broader context of P. amoebophila's metabolic network, particularly its relationship to nutrient acquisition and energy metabolism during different growth phases.

• Development of specific inhibitors: Identifying unique structural features of P. amoebophila isoleucine-tRNA ligase could guide the development of specific inhibitors with potential applications in understanding chlamydial biology.

• Modified nucleoside incorporation: Investigating how modified nucleosides in tRNA affect recognition and aminoacylation by isoleucine-tRNA ligase could provide insights into translational regulation mechanisms .

These research directions would not only advance our understanding of P. amoebophila biology but also contribute to broader knowledge about evolutionary adaptation of essential translational machinery.

How might findings from Protochlamydia amoebophila isoleucine-tRNA ligase research inform our understanding of bacterial evolution and host adaptation?

Research on P. amoebophila isoleucine-tRNA ligase offers valuable perspectives on bacterial evolution and host adaptation:

• Evolutionary diversification: Comparative analysis of aminoacyl-tRNA synthetases across the chlamydial lineage provides a window into the evolutionary divergence of these ancient enzymes. The differences in tryptophan content between P. amoebophila and Chlamydiaceae isoleucine-tRNA ligases reflect distinct evolutionary trajectories following adaptation to different host environments .

• Host-driven selection pressures: The absence of up-Trp selection in P. amoebophila isoleucine-tRNA ligase, contrasting with patterns observed in mammalian-infecting chlamydial species, suggests that host immune responses creating tryptophan-limited environments have been a significant driver of protein evolution in mammalian pathogens but not in protozoan symbionts .

• Molecular clock applications: The degree of sequence divergence in highly conserved proteins like isoleucine-tRNA ligase can be used to estimate divergence times between chlamydial lineages, providing insights into the timeline of adaptation to different host environments.

• Co-evolutionary dynamics: Patterns of amino acid usage in isoleucine-tRNA ligase and other essential proteins may reflect co-evolutionary dynamics between chlamydial species and their hosts, particularly regarding nutrient availability and immune pressures.

• Evolutionary trade-offs: The maintenance of editing activity against potential translation errors must be balanced against the metabolic cost of enzyme production and the risk of tryptophan-dependent translational regulation, providing a model system for studying evolutionary trade-offs in essential cellular machinery.