Recombinant Protochlamydia amoebophila Ribosomal protein S12 methylthiotransferase RimO (rimO)

What is the function of Protochlamydia amoebophila RimO?

Protochlamydia amoebophila RimO belongs to the family of radical-S-adenosylmethionine enzymes that catalyze the methylthiolation of ribosomal protein S12. Specifically, it methylthiolates residue D88 in the S12 protein, creating a unique post-translational modification that affects ribosomal function . This enzyme represents an important component of bacterial protein modification systems and shares strong sequence similarity with MiaB, which performs analogous modifications but on tRNA rather than proteins . The functional activity of RimO is particularly significant given that P. amoebophila is an intracellular bacterium that has evolved specialized mechanisms for energy acquisition and cellular processes within host cells .

How does P. amoebophila RimO differ from RimO enzymes in other bacterial species?

While the specific differences between P. amoebophila RimO and other bacterial RimO variants are not extensively documented in the provided search results, we can infer potential differences based on the unique characteristics of P. amoebophila as an intracellular pathogen. The RimO enzyme belongs to an ancient family of methylthiotransferases with four major subgroups, with RimO being unique among these in its modification of proteins rather than RNA . Despite this functional difference in substrate specificity, RimO has not significantly diverged at the sequence level from other methylthiotransferases, even within the C-terminal TRAM domain that is presumed to be responsible for substrate binding and recognition . This conservation suggests fundamental structural similarities across different bacterial species while potentially allowing for species-specific adaptations in substrate recognition.

What expression systems are most effective for producing recombinant P. amoebophila RimO?

Based on analogous research with other Protochlamydia proteins, E. coli expression systems have proven effective for producing recombinant chlamydial proteins. For instance, researchers successfully expressed and purified the PamNTT1 ATP/ADP transporter from P. amoebophila using heterologous expression methods . For RimO specifically, expression systems that accommodate radical SAM enzymes are recommended, including:

When expressing RimO, anaerobic conditions are typically necessary to preserve the Fe-S clusters intrinsic to radical SAM enzymes, with purification performed in an anaerobic chamber to maintain enzyme activity .

What are the optimal conditions for assessing P. amoebophila RimO methylthiotransferase activity in vitro?

To assess methylthiotransferase activity of P. amoebophila RimO, researchers should establish an in vitro assay system based on the following parameters:

• Buffer composition: 50 mM HEPES pH 7.5, 100 mM KCl, 5% glycerol

• Required cofactors:

  • S-adenosylmethionine (SAM) as methyl donor

  • Dithionite as reducing agent

  • Fe-S cluster reconstitution components (FeCl₃, Na₂S)

• Substrate preparation: Purified recombinant S12 ribosomal protein (preferably from P. amoebophila)

• Reaction conditions: Anaerobic environment, 37°C, 60-minute incubation

• Activity detection:

  • Mass spectrometry to detect methylthiolation at D88 residue

  • Radioactive assay using ¹⁴C-SAM to track methyl transfer

The critical factors influencing activity include maintaining anaerobic conditions to preserve the Fe-S clusters, ensuring complete reconstitution of the enzyme with its Fe-S cofactors, and using freshly prepared reduction systems . Based on analogous enzymes, the reaction likely requires careful optimization of the ratio between enzyme, substrate, and cofactors to achieve detectable activity levels.

How can researchers effectively purify recombinant P. amoebophila RimO while maintaining its activity?

Purifying active recombinant P. amoebophila RimO requires specific strategies to preserve the radical SAM enzyme's activity:

• Expression optimization:

  • Induce expression at lower temperatures (16-18°C)

  • Include iron and cysteine supplements in growth media

  • Express under microaerobic or anaerobic conditions

• Purification protocol:

  • All steps performed anaerobically or with oxygen-scavenging systems

  • Buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Addition of protease inhibitors to prevent degradation

• Recommended purification scheme:

• Fe-S cluster reconstitution: Critical for activity; performed by incubating purified protein with FeCl₃, Na₂S, and DTT under anaerobic conditions followed by desalting to remove excess reconstitution components .

Drawing from successful purification of other P. amoebophila proteins like PamNTT1 , maintaining protein stability throughout the purification process is essential. The addition of glycerol and reducing agents in all buffers helps preserve enzyme structure and activity.

How does the structure-function relationship of P. amoebophila RimO compare to other methylthiotransferases?

P. amoebophila RimO represents a fascinating case of evolutionary adaptation within the methylthiotransferase family. Despite RimO targeting protein substrates while related enzymes like MiaB modify tRNA, these enzymes show remarkable sequence conservation, particularly in the C-terminal TRAM domain . This domain in the RumA methyltransferase is known to bind RNA substrates, suggesting that RimO may have evolved altered substrate specificity while maintaining structural elements .

Structural analysis predictions based on related methylthiotransferases indicate:

• Domain organization:

  • N-terminal radical SAM domain with coordinated [4Fe-4S] cluster

  • Central domain containing additional Fe-S clusters

  • C-terminal TRAM domain for substrate recognition

• Key functional elements:

  • Radical SAM motif (CxxxCxxC) for [4Fe-4S] cluster binding

  • SAM binding pocket within the radical SAM domain

  • Substrate binding residues in the TRAM domain

This represents a remarkable example of how enzymes can evolve new specificities (protein vs. RNA) without significant divergence at the sequence level, making RimO and MiaB "the most extreme known case of resemblance between enzymes modifying protein and nucleic acid" .

What are the methodological challenges in studying the in vivo effects of RimO methylthiolation in P. amoebophila?

Investigating the in vivo effects of RimO methylthiolation in P. amoebophila presents significant methodological challenges due to several factors:

• Intracellular lifestyle limitations:

  • P. amoebophila is an obligate intracellular bacterium, complicating genetic manipulation

  • Requires host amoeba cells for growth and development

  • Energy parasitism mechanisms make metabolic studies complex

• Genetic system constraints:

  • Limited genetic tools available for P. amoebophila

  • Challenges in creating knockout or point mutants to study RimO function

• Experimental approaches:

• Developmental cycle considerations:
  Similar to other Chlamydiae, P. amoebophila likely undergoes a biphasic developmental cycle with distinct elementary bodies and reticulate bodies. Studies must consider which developmental stage is being examined, as protein expression and modification may vary between stages .

How can researchers investigate potential cross-talk between RimO-mediated S12 methylthiolation and other post-translational modifications in P. amoebophila?

Investigation of potential cross-talk between RimO-mediated methylthiolation and other post-translational modifications (PTMs) requires sophisticated experimental approaches:

• Mass spectrometry-based PTM mapping:

  • Employ advanced proteomics to identify the complete set of PTMs on S12 protein

  • Use sequential enrichment strategies to detect co-occurring modifications

  • Apply quantitative proteomics to measure stoichiometry of different modifications

• Timing analysis of modifications:

  • Pulse-chase experiments with isotope-labeled amino acids

  • Time-course sampling during P. amoebophila developmental cycle

  • Correlation of modification patterns with specific bacterial life stages

• Reconstituted in vitro systems:

  • Sequential addition of purified modification enzymes to determine order effects

  • Competition assays between different modifying enzymes

  • Site-directed mutagenesis of surrounding residues to assess context effects

• Bioinformatic prediction of modification interactions:
  Analyze sequence conservation patterns around the D88 residue to identify potential "modification codes" that might regulate cross-talk between different PTMs.

These studies are particularly relevant given that P. amoebophila, as an energy parasite, has evolved specialized mechanisms for ATP/ADP transport , suggesting that ribosomal regulation through post-translational modifications may play a crucial role in its adaptation to intracellular life.

How does our understanding of P. amoebophila RimO contribute to broader knowledge of bacterial adaptation strategies?

The study of P. amoebophila RimO provides critical insights into bacterial adaptation strategies, particularly for intracellular pathogens:

• Evolutionary conservation:
  The remarkable conservation of RimO structure despite substrate switching from RNA to protein suggests fundamental constraints on enzyme evolution while highlighting the plasticity of substrate recognition mechanisms . This exemplifies how bacteria can repurpose existing enzymatic machinery for new functions during adaptation.

• Specialized ribosomal modifications:
  The methylthiolation of S12 likely represents a specialized adaptation affecting translational fidelity or efficiency. As P. amoebophila is an energy parasite that exploits host ATP , precise control of protein synthesis through ribosomal modifications may be critical for its survival strategy.

• Intracellular lifestyle adaptations:
  Like other Chlamydiae, P. amoebophila synthesizes a specialized peptidoglycan ring during division that differs from conventional bacterial cell walls . The presence of specific ribosomal modifications may similarly reflect specialized adaptations to the intracellular environment.

Understanding these adaptation mechanisms provides broader perspectives on bacterial evolution and host-pathogen interactions, with potential applications for studying related human pathogens in the Chlamydiales order.

What are the most promising approaches for determining the structural basis of substrate recognition by P. amoebophila RimO?

Several complementary approaches hold promise for elucidating the structural basis of substrate recognition by P. amoebophila RimO:

• X-ray crystallography:

  • Co-crystallization of RimO with S12 substrate or substrate analogs

  • Use of non-hydrolyzable SAM analogs to capture the enzyme-substrate complex

  • Resolution requirements: 2.5 Å or better to visualize key interactions

• Cryo-electron microscopy:

  • Analysis of RimO bound to whole ribosomal subunits

  • Advantage of visualizing the natural substrate environment

  • Potential for capturing multiple binding states

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps protein dynamics and binding interfaces

  • Can identify conformational changes upon substrate binding

  • Less resource-intensive than crystallography or cryo-EM

• Computational approaches:

• Site-directed mutagenesis coupled with activity assays:

  • Systematic mutation of TRAM domain residues

  • Identification of key residues for protein vs. RNA substrate discrimination

  • Correlation of structural predictions with functional outcomes

The heterologous expression and purification approach that proved successful for PamNTT1 could provide the foundation for obtaining sufficient quantities of P. amoebophila RimO for these structural studies.

What techniques can researchers employ to investigate the impact of S12 methylthiolation on ribosomal function and translational fidelity in P. amoebophila?

To investigate the impact of S12 methylthiolation on ribosomal function and translational fidelity, researchers can employ these advanced techniques:

• In vitro translation systems:

  • Reconstituted translation assays using purified components

  • Comparison of ribosomes with methylthiolated vs. non-modified S12

  • Measurement of translation rate, fidelity, and response to antibiotics

• Ribosome profiling:

  • Next-generation sequencing of ribosome-protected mRNA fragments

  • Analysis of ribosome positioning and pausing

  • Comparison between wild-type and RimO-deficient strains (if genetic systems permit)

• Single-molecule fluorescence methods:

  • Real-time observation of translation by individual ribosomes

  • Direct measurement of elongation rates and miscoding events

  • Correlation of translational dynamics with S12 modification status

• Structural analysis of the ribosome:

  • Cryo-EM structures of ribosomes with modified S12

  • Examination of conformational changes induced by methylthiolation

  • Correlation with functional studies

• Differential proteomics:

  • Quantitative comparison of proteomes in the presence/absence of RimO

  • Identification of proteins whose expression is most affected by S12 methylthiolation

  • Analysis of codon usage bias in affected genes

These approaches could reveal how this specialized post-translational modification contributes to P. amoebophila's adaptation to its intracellular lifestyle, where energy parasitism through ATP/ADP transport proteins plays a crucial role in survival .

How can researchers address issues with expression and solubility of recombinant P. amoebophila RimO?

Researchers often encounter challenges with expression and solubility when working with recombinant methylthiotransferases. Based on successful approaches with other P. amoebophila proteins , consider these solutions:

• Expression optimization strategies:

• Solubility enhancement approaches:

  • Reduce expression temperature to 16-18°C

  • Include stabilizing agents (glycerol, arginine, trehalose) in lysis buffer

  • Screen various detergents at low concentrations to improve solubility

  • Test different buffer systems (HEPES, phosphate, Tris) at various pH values (7.0-8.5)

• Fe-S cluster considerations:

  • Add iron and cysteine supplements to growth media

  • Include iron chelators during purification to prevent oxidative damage

  • Perform all steps under anaerobic conditions when possible

• Refolding protocols:
  If inclusion bodies persist, develop a refolding strategy using gradual dialysis with decreasing concentrations of mild denaturants coupled with Fe-S cluster reconstitution.

What controls and validation steps are essential when studying P. amoebophila RimO activity?

Rigorous controls and validation steps are critical when studying the enzyme activity of P. amoebophila RimO:

• Essential controls for activity assays:

  • Negative control: Reaction without enzyme

  • Substrate specificity control: Non-S12 ribosomal proteins

  • Active site mutant control: SAM-binding site mutation

  • Fe-S cluster dependence: Reaction with oxidized/damaged enzyme

• Validation of methylthiolation:

  • Mass spectrometry confirmation of exact modification site (D88)

  • Multiple reaction monitoring (MRM) for quantitative assessment

  • Antibodies raised against the methylthiolated peptide for immunoblotting

  • Chemical stability tests to differentiate methylthiolation from other modifications

• Protein quality assessment:

• Reproducibility considerations:

  • Prepare enzyme fresh for each experiment or validate stability during storage

  • Standardize anaerobic conditions using oxygen indicators

  • Include internal standards for quantitative measurements

  • Perform assays with at least three independent enzyme preparations

These validation steps are particularly important given that RimO represents a unique case of an enzyme that modifies protein rather than RNA despite strong sequence conservation with RNA-modifying enzymes .

How can structural insights from P. amoebophila RimO inform antibiotic development against intracellular pathogens?

Structural studies of P. amoebophila RimO can provide valuable insights for antibiotic development against intracellular pathogens, particularly those in the Chlamydiales order:

• Novel drug target potential:

  • Ribosomal protein modifications represent underexplored targets for antibiotics

  • The essential nature of protein synthesis makes ribosome-associated enzymes attractive targets

  • Substrate-binding specificity of RimO offers potential for selective inhibition

• Structure-based drug design approaches:

  • Virtual screening against the substrate binding pocket of RimO

  • Fragment-based drug discovery using structural data

  • Rational design of analogs that compete with S12 binding

• Exploiting unique features of intracellular pathogens:

  • Target the energy parasitism mechanisms used by intracellular bacteria

  • Design compounds that disrupt both RimO function and ATP/ADP transport

  • Develop dual-action inhibitors affecting both ribosomal modification and cell division processes

• Translational implications:

  • Insights from P. amoebophila RimO could inform treatments for related human pathogens

  • The narrow peptidoglycan ring structure in Chlamydiales combined with ribosomal modification targeting could provide synergistic treatment approaches

  • Potential applications for treating resistant infections through novel mechanisms of action

The study of RimO contributes to understanding fundamental bacterial adaptation mechanisms that could reveal vulnerabilities exploitable for therapeutic development.

What is the evolutionary significance of the conservation between protein-modifying RimO and RNA-modifying MiaB enzymes?

The remarkable conservation between RimO and MiaB enzymes despite their different substrates represents an intriguing case study in molecular evolution:

• Evolutionary hypotheses:

  • Common ancestor with substrate ambiguity that subsequently specialized

  • Convergent evolution constrained by the chemistry of the methylthiolation reaction

  • Functional co-option of an RNA-modifying enzyme for protein modification

• Mechanistic implications:
  The conservation of the TRAM domain between RimO and RNA-modifying enzymes suggests a fundamental similarity in how these enzymes recognize structurally different substrates . This conservation indicates that certain protein structural elements may mimic RNA structural features recognized by the ancestral enzyme.

• Comparative analysis framework:

• Broader significance:
  This system exemplifies how evolution can repurpose existing enzymatic machinery for new functions while maintaining core mechanistic features. The case of RimO and MiaB represents "the most extreme known case of resemblance between enzymes modifying protein and nucleic acid" , providing insights into the molecular plasticity that drives bacterial adaptation and evolution.

Understanding this evolutionary relationship could inform research into other enzyme families and reveal principles of molecular evolution applicable across biological systems.