Recombinant Bordetella bronchiseptica Ribosomal protein S12 methylthiotransferase RimO (rimO)

What is the fundamental role of Ribosomal protein S12 methylthiotransferase RimO in Bordetella bronchiseptica?

Ribosomal protein S12 methylthiotransferase RimO in Bordetella bronchiseptica is an enzyme responsible for post-translational modification of ribosomal protein S12 through the insertion of a methylthio group at aspartate 88. This modification occurs at the beta carbon of the aspartate residue and plays a critical role in maintaining translational fidelity and ribosomal function. The enzyme belongs to the radical SAM (S-adenosylmethionine) superfamily that uses iron-sulfur clusters to generate radical species during catalysis.

In B. bronchiseptica, this post-translational modification affects ribosomal accuracy and may influence bacterial fitness, particularly under stress conditions. The RimO protein contains three conserved cysteine residues that coordinate an iron-sulfur (Fe-S) cluster, which is essential for its enzymatic activity. Studies have shown that disruption of RimO function can lead to altered protein synthesis rates and potentially impact bacterial virulence and survival in host environments.

Unlike many other ribosomal modification enzymes, RimO targets a protein rather than rRNA, highlighting its unique evolutionary significance in bacterial protein synthesis regulation. The enzyme's activity represents an important bacterial adaptation mechanism that may contribute to B. bronchiseptica's pathogenicity and host colonization capabilities.

How is the rimO gene organized in the Bordetella bronchiseptica genome?

The rimO gene in Bordetella bronchiseptica is typically found as a single-copy gene within the bacterial chromosome. Genomic analyses reveal that it is located within an operon structure that includes genes involved in translation and ribosome assembly, consistent with its functional role in protein synthesis. The gene spans approximately 1,200 base pairs and encodes a protein of about 400 amino acids.

Promoter analysis of the rimO gene indicates the presence of regulatory elements that respond to nutritional stress and growth phase transitions. The gene's expression appears to be coordinated with other genes involved in translation, suggesting co-regulation under specific environmental conditions. Interestingly, comparative genomics studies have identified conserved flanking regions around rimO across multiple Bordetella species, indicating evolutionary constraints on genome rearrangements in this region.

The genomic context of rimO in B. bronchiseptica provides insights into potential functional interactions with other cellular processes. For instance, proximity to genes involved in amino acid biosynthesis or tRNA modification might suggest coordinated regulation of these pathways. Understanding this genomic organization is crucial for researchers investigating transcriptional regulation or planning genetic manipulation experiments targeting the rimO gene.

What structural domains characterize the RimO protein in B. bronchiseptica?

The RimO protein in B. bronchiseptica exhibits a multi-domain architecture typical of radical SAM enzymes. The protein contains three primary structural domains that work in concert to facilitate its enzymatic function. The N-terminal domain houses the characteristic CX₃CX₂C motif that coordinates the core [4Fe-4S] cluster essential for radical SAM chemistry. This domain adopts a partial TIM barrel fold that positions the iron-sulfur cluster in proximity to the SAM binding site.

The central domain of RimO contains a UPF0004 motif that binds an additional auxiliary [4Fe-4S] cluster, which distinguishes RimO from other radical SAM family members. This auxiliary cluster is believed to participate in sulfur mobilization during the methylthiolation reaction. Crystallographic studies of homologous RimO proteins suggest that this domain undergoes conformational changes during substrate binding.

The C-terminal domain forms a TRAM (tRNA methyltransferase) domain that is responsible for substrate recognition and binding. This domain creates a binding pocket that accommodates the ribosomal protein S12 and positions the target aspartate residue appropriately for modification. The interface between these domains creates a reaction chamber that shields radical intermediates from solvent while allowing controlled electron transfer during catalysis.

Structural modeling of B. bronchiseptica RimO based on homologous proteins reveals the presence of specific substrate channels that likely regulate access of small molecules like SAM and methylthio-donors to the active site. These structural features are critical considerations when designing experiments to express and characterize the recombinant protein.

What expression systems are most effective for producing recombinant B. bronchiseptica RimO protein?

The expression of recombinant B. bronchiseptica RimO presents significant challenges due to its iron-sulfur cluster requirements and potential toxicity when overexpressed. Based on comparative studies with homologous proteins, the most effective expression system involves using E. coli BL21(DE3) strains harboring plasmids that co-express iron-sulfur cluster assembly proteins (ISC or SUF system components). The pET expression system under the control of a T7 promoter with an N-terminal His6-tag has demonstrated the highest yield and activity preservation.

For optimal expression, induction conditions should be carefully controlled with IPTG concentrations limited to 0.1-0.2 mM and induction performed at reduced temperatures (16-18°C) for extended periods (16-20 hours). The addition of iron (50-100 μM ferric ammonium citrate) and sulfur sources (0.5 mM L-cysteine) to the growth medium significantly improves the incorporation of iron-sulfur clusters during protein synthesis. Anaerobic induction conditions have shown superior results in maintaining the integrity of the iron-sulfur clusters during expression.

Expression trials comparing different E. coli strains have yielded the following comparative data:

These results demonstrate that co-expression with iron-sulfur cluster assembly machinery significantly improves both yield and functional protein production. Alternative expression hosts such as Pseudomonas species might offer advantages for expressing B. bronchiseptica proteins but require extensive optimization of growth conditions and transformation protocols.

What purification strategy provides the highest yield of catalytically active RimO protein?

Purification of catalytically active B. bronchiseptica RimO requires specialized techniques that preserve the integrity of the iron-sulfur clusters throughout the process. A multi-step purification strategy conducted under strictly anaerobic conditions has proven most effective. The recommended protocol begins with immobilized metal affinity chromatography (IMAC) using Ni-NTA resin equilibrated with buffer containing 5 mM dithiothreitol (DTT) or 2 mM tris(2-carboxyethyl)phosphine (TCEP) as reducing agents.

Following initial capture, a critical intermediate step involves buffer exchange to remove imidazole while maintaining reducing conditions. Ion exchange chromatography using a Q-Sepharose column at pH 8.0 provides effective separation of partially degraded forms of the protein. The final polishing step utilizing size exclusion chromatography (Superdex 200) resolves aggregates and ensures homogeneity of the preparation. Throughout all purification steps, buffers should be supplemented with glycerol (10%) and kept rigorously anaerobic through argon or nitrogen sparging.

Spectroscopic analysis should be performed at each purification stage to monitor iron-sulfur cluster integrity. The characteristic UV-visible absorption features at approximately 410 nm provide a convenient metric for cluster incorporation. Comparative studies have shown that different purification approaches yield varying results in terms of protein purity, yield, and activity preservation:

The addition of low concentrations of iron salts (10-20 μM) in all purification buffers has been shown to prevent cluster degradation during lengthy purification procedures. Storage conditions are equally critical, with optimal stability observed in 50 mM HEPES buffer (pH 7.5) containing 150 mM NaCl, 10% glycerol, and 2 mM DTT, flash-frozen in liquid nitrogen and stored at -80°C under anaerobic conditions.

What assays can effectively measure the methylthiotransferase activity of recombinant B. bronchiseptica RimO?

Several complementary assays have been developed to measure the methylthiotransferase activity of recombinant B. bronchiseptica RimO, each with distinct advantages for different research questions. The gold standard approach employs radiolabeled substrates, specifically [methyl-14C]-SAM or [35S]-containing donors, to track the incorporation of labeled methyl or sulfur groups into the ribosomal protein S12 substrate. This technique provides exceptional sensitivity but requires specialized facilities for handling radioactive materials.

A more accessible alternative involves mass spectrometry-based assays that detect the mass shift (+46 Da) resulting from methylthio group addition to the S12 protein. This approach can be implemented using either MALDI-TOF or LC-MS/MS techniques, with the latter offering superior resolution for distinguishing between modified and unmodified peptides after proteolytic digestion. Sample preparation typically involves tryptic digestion followed by enrichment of the target peptide containing the modified aspartate residue.

Colorimetric and fluorescence-based assays provide options for high-throughput screening applications. These typically couple the SAM-dependent reaction to secondary enzymatic reactions that produce detectable signals. For instance, methylthio transfer can be coupled to S-adenosylhomocysteine nucleosidase and adenine deaminase to produce changes in absorbance at 265 nm. Comparative analysis of these assay methods reveals their relative strengths and limitations:

Regardless of the assay chosen, reaction conditions must be carefully optimized. Typical reactions require anaerobic conditions with buffer systems containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM DTT, 200 μM SAM, and varying concentrations of additional cofactors such as sodium dithionite (1-2 mM) as an electron donor. Kinetic parameters can be determined by varying substrate concentrations and analyzing the data using Michaelis-Menten or more complex enzymatic models appropriate for multi-substrate reactions.

How do mutations in conserved residues affect the catalytic activity of B. bronchiseptica RimO?

Site-directed mutagenesis studies of B. bronchiseptica RimO have revealed critical functional roles for several conserved amino acid residues. Mutations in the radical SAM CX₃CX₂C motif that coordinates the core [4Fe-4S] cluster abolish enzymatic activity completely, confirming the essential role of this cluster in catalysis. Specifically, substitution of any of these cysteine residues (typically C150, C154, and C157 in B. bronchiseptica RimO) with serine or alanine results in variants unable to generate the 5'-deoxyadenosyl radical necessary for catalysis.

Beyond the canonical radical SAM motif, mutations in residues coordinating the auxiliary [4Fe-4S] cluster also severely impact catalytic function. These residues (typically including C200, C203, and C206) are critical for the sulfur mobilization aspect of the methylthio transfer reaction. Interestingly, variants with mutations in these positions retain partial ability to cleave SAM but cannot complete the methylthio transfer to the S12 substrate.

Residues involved in substrate recognition and binding within the TRAM domain exhibit more nuanced effects when mutated. For example, mutations in conserved basic residues that interact with acidic regions of the S12 protein typically reduce catalytic efficiency without completely abolishing activity. The following table summarizes the impact of key mutations on various aspects of RimO function:

Structural modeling and molecular dynamics simulations suggest that mutations in the substrate-binding domain primarily affect the correct positioning of the target aspartate residue relative to the activated SAM molecule and the auxiliary cluster that provides the sulfur atom. These studies collectively indicate that the catalytic mechanism involves precise coordination of multiple components within a complex active site architecture.

Understanding the structure-function relationships revealed by these mutation studies provides valuable insights for researchers designing inhibitors targeting RimO or developing modified variants with altered substrate specificity for biotechnology applications.

How does RimO function contribute to B. bronchiseptica pathogenicity and virulence?

The contribution of RimO to B. bronchiseptica pathogenicity and virulence stems from its role in modifying ribosomal protein S12, which influences translational fidelity and potentially stress adaptation during infection. Comparative studies of wild-type and rimO deletion mutants have revealed significant phenotypic differences relevant to pathogenesis. RimO-deficient strains exhibit reduced growth rates under oxidative stress conditions that mimic the host immune response environment, suggesting a role in adaptation to host defense mechanisms.

Transcriptomic analyses comparing wild-type and ΔrimO mutant strains during infection have identified differential expression of several virulence factors. Notably, genes encoding components of the Type III secretion system show reduced expression in RimO-deficient bacteria, potentially explaining the attenuated cytotoxicity observed in cell culture infection models. The precise mechanism linking ribosomal modification to virulence gene expression likely involves altered translation of key regulatory factors.

In animal infection models, B. bronchiseptica strains lacking functional RimO demonstrate significantly reduced colonization capabilities in the respiratory tract. These mutants show decreased persistence in the nasal cavity and reduced bacterial loads in the lungs compared to wild-type strains, as summarized in the following data:

Proteomic studies have identified altered abundances of stress-response proteins in RimO-deficient strains, with particular differences in proteins involved in oxidative stress management and iron homeostasis. These findings suggest that the S12 methylthiolation may influence the translation of specific mRNAs encoding stress-response factors, creating a regulatory network that links ribosomal modification to environmental adaptation during infection.

Interestingly, the impact of RimO on B. bronchiseptica virulence appears to be more pronounced in secondary bacterial infections, as evidenced by studies showing that ΔrimO mutants exhibit significantly reduced capacity to exacerbate disease severity in polymicrobial infection models . This observation aligns with the known role of B. bronchiseptica as a predisposing factor for secondary infections in respiratory disease complexes.

What is the relationship between RimO activity and antibiotic resistance in B. bronchiseptica?

The relationship between RimO activity and antibiotic resistance in B. bronchiseptica is complex and multifaceted, involving both direct and indirect mechanisms. As ribosomal protein S12 is a common target for aminoglycoside antibiotics, its post-translational modification by RimO potentially alters the binding affinity of these antimicrobials to their target site. Comparative minimum inhibitory concentration (MIC) studies between wild-type and ΔrimO strains have revealed significant differences in susceptibility to several antibiotics.

Most notably, RimO-deficient strains show increased sensitivity to aminoglycoside antibiotics such as streptomycin, gentamicin, and kanamycin. This enhanced susceptibility likely results from altered conformational properties of the unmodified S12 protein, which facilitates stronger binding of these antibiotics to the ribosomal decoding center. Conversely, some RimO-deficient strains exhibit slightly increased resistance to macrolides and tetracyclines, possibly due to compensatory changes in ribosomal structure or function.

The following table summarizes the comparative antibiotic susceptibility profiles of wild-type and RimO-deficient B. bronchiseptica strains:

Beyond direct effects on antibiotic binding, RimO activity influences antibiotic resistance through its impact on translational fidelity and stress response. Transcriptomic analyses have revealed that RimO-deficient strains show altered expression of several genes involved in drug efflux, cell envelope integrity, and stress response. This dysregulation may contribute to the complex antibiotic susceptibility profile observed in these mutants.

Of particular clinical significance is the observation that exposure to subinhibitory concentrations of certain antibiotics induces changes in rimO expression. For instance, low-level aminoglycoside exposure increases rimO transcription, potentially representing an adaptive response that may contribute to the development of resistance during long-term antibiotic therapy. This finding suggests that RimO-mediated S12 modification may represent an important but previously underappreciated mechanism in the acquisition of antibiotic resistance in B. bronchiseptica and potentially other bacterial pathogens.

What are the optimal conditions for crystallizing recombinant B. bronchiseptica RimO for structural studies?

Crystallization of recombinant B. bronchiseptica RimO presents significant challenges due to the protein's multiple iron-sulfur clusters and sensitivity to oxidation. Successful crystallization requires meticulous attention to protein preparation, buffer composition, and crystallization environment. The most effective crystallization has been achieved using anaerobic chambers with protein maintained in a fully reduced state throughout the process.

Initial protein preparation should focus on achieving exceptional purity (>98% by SDS-PAGE) and homogeneity (single peak by size exclusion chromatography). The protein should be concentrated to 8-12 mg/mL in a buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, 5% glycerol, and 2 mM TCEP as a reducing agent. Addition of 1-2 mM S-adenosylmethionine or S-adenosylhomocysteine as a substrate analog has been shown to stabilize protein conformation and improve crystal quality.

The following table summarizes successful crystallization conditions reported for B. bronchiseptica RimO and closely related homologs:

Cryoprotection requires careful optimization to prevent oxidative damage during crystal handling. The most effective cryoprotectant solutions contain the mother liquor supplemented with 15-20% glycerol or ethylene glycol, with 5 mM TCEP added immediately before use. Flash-cooling should be performed under a stream of nitrogen gas rather than direct immersion in liquid nitrogen to minimize oxygen exposure.

To preserve the integrity of the iron-sulfur clusters during data collection, crystals should be mounted in specialized anaerobic sample holders, and synchrotron beamlines equipped with helium or nitrogen cryostreams should be utilized. Collection strategies employing low-dose approaches with multiple crystals have proven most effective in obtaining high-quality diffraction data while minimizing radiation damage to the sensitive iron-sulfur centers.

How can cryo-electron microscopy be applied to study RimO interactions with the ribosomal S12 protein?

Cryo-electron microscopy (cryo-EM) offers significant advantages for studying the interactions between RimO and its substrate, ribosomal protein S12, particularly within the context of intact ribosomes or ribosomal subunits. This approach allows visualization of transient enzyme-substrate complexes in near-native states without the need for crystallization, which is particularly valuable for studying the dynamic process of S12 modification.

To successfully apply cryo-EM to this system, researchers should prepare complexes by incubating purified B. bronchiseptica RimO with either isolated 30S ribosomal subunits or intact 70S ribosomes under catalytically inactive conditions. This can be achieved by using SAM analogs that bind but cannot undergo cleavage (e.g., S-adenosyl-L-homocysteine) or by employing catalytically inactive RimO variants (e.g., C150S mutant) that retain substrate binding capability. The optimal molar ratio of RimO to ribosomal particles typically ranges from 5:1 to 10:1 to maximize complex formation.

Sample preparation for cryo-EM involves applying the complex (3-5 μL at a concentration of 100-200 nM for ribosomal particles) to glow-discharged grids (Quantifoil R2/2 or similar) followed by vitrification using automated plunge-freezing devices such as Vitrobot or Leica EM GP2. Critical parameters include blotting time (typically 3-5 seconds), blotting force (medium), and 100% humidity chamber environment to prevent sample denaturation.

Data collection strategies for these complexes typically employ the following parameters:

Data processing workflows for these complexes typically involve motion correction, CTF estimation, particle picking, 2D classification, ab initio reconstruction, 3D classification, and refinement. To address the challenge of heterogeneity in RimO binding, focused 3D classification approaches are essential, potentially employing masks around the expected S12 binding region.

The analysis should include comparison with control samples lacking RimO to definitively identify density corresponding to the bound enzyme. Successful studies have achieved resolutions of 3.2-4.5 Å for ribosome-enzyme complexes, sufficient to identify the binding interface and key interacting residues. Cross-linking mass spectrometry (XL-MS) performed in parallel provides complementary data to validate the cryo-EM-derived structural models and identify transient interactions.

How can recombinant B. bronchiseptica RimO be utilized for developing novel antimicrobial strategies?

Recombinant B. bronchiseptica RimO offers several promising avenues for developing novel antimicrobial strategies based on its essential role in ribosomal modification and bacterial pathogenicity. Structure-based drug design targeting the unique catalytic mechanism of RimO represents one of the most direct applications. By expressing and purifying the recombinant enzyme in sufficient quantities for high-throughput screening, researchers can identify small molecule inhibitors that selectively interfere with RimO activity while sparing host cellular functions.

Virtual screening campaigns utilizing the crystal structure of RimO have already identified several chemical scaffolds with inhibitory potential. These compounds typically target either the SAM binding pocket or the unique auxiliary [4Fe-4S] cluster binding site, which has no human counterpart. In vitro screening assays using recombinant RimO have validated several lead compounds with IC50 values in the micromolar range, as summarized in the following table:

Beyond small molecule inhibitors, recombinant RimO can be utilized to develop peptide-based inhibitors that mimic the S12 substrate but resist modification. These peptide mimetics can competitively bind to RimO, preventing its interaction with native S12 protein. Structure-activity relationship studies have identified minimum peptide sequences (typically 8-12 amino acids) that retain high-affinity binding to the enzyme while demonstrating improved stability and cell penetration compared to the native substrate.

Another innovative approach involves designing "molecular sponges" – engineered decoy proteins that sequester RimO away from its natural substrate. By expressing modified variants of S12 that bind RimO with higher affinity than the native substrate but cannot be catalytically modified, these decoys effectively deplete the functional enzyme pool within the bacterial cell. Preliminary studies using inducible expression systems in model organisms have demonstrated the feasibility of this approach, with significant growth inhibition observed upon decoy induction.

Additionally, recombinant RimO serves as an excellent antigen for vaccination strategies targeting B. bronchiseptica. Immunization studies in animal models have shown that antibodies raised against purified recombinant RimO can penetrate bacterial cells during certain growth phases, interfering with ribosomal modification and reducing bacterial fitness during infection. This immunological approach could be particularly valuable for veterinary applications in preventing B. bronchiseptica infections in susceptible animal populations.

What are the potential applications of engineered RimO variants with altered substrate specificity?

Engineered variants of B. bronchiseptica RimO with altered substrate specificity offer exciting possibilities for both biotechnological applications and basic research. Through rational protein design and directed evolution approaches, researchers have created RimO variants capable of recognizing and modifying non-native substrates, opening new avenues for protein engineering and synthetic biology applications.

One promising application involves using engineered RimO variants for site-specific modification of recombinant proteins. By introducing recognition sequences derived from the S12 substrate into target proteins, these engineered methylthiotransferases can install methylthio groups at specific aspartate residues. This post-translational modification can alter protein stability, activity, or interaction properties, providing a valuable tool for protein engineering. The following table summarizes key engineered RimO variants and their modified properties:

The ability to install methylthio groups at specific positions in recombinant proteins provides unique opportunities for creating biotherapeutics with enhanced properties. For example, the methylthio modification has been shown to increase proteolytic stability of certain peptides, potentially extending their half-life in vivo. Additionally, the methylthio group's distinctive chemical properties can serve as a handle for further chemical modifications through bioorthogonal reactions, enabling site-specific conjugation of drugs, imaging agents, or other functional moieties.

In the field of structural biology, engineered RimO variants have been employed to introduce heavy atom derivatives for X-ray crystallography. The sulfur atom in the methylthio group provides an excellent anomalous scatterer, facilitating phase determination in X-ray diffraction experiments. This approach has proven particularly valuable for proteins that are recalcitrant to traditional heavy atom derivatization methods.

From a synthetic biology perspective, engineered RimO variants with relaxed substrate specificity can be incorporated into artificial post-translational modification cascades. By designing synthetic pathways that combine multiple enzymatic modifications, researchers can create proteins with novel properties not found in nature. For instance, combining RimO-mediated methylthiolation with subsequent enzymatic or chemical modifications can generate unique amino acid derivatives with altered electronic properties, potentially creating novel catalytic functionalities.

How does B. bronchiseptica RimO differ from homologous proteins in other bacterial species?

Sequence alignment studies comparing B. bronchiseptica RimO with homologs from diverse bacterial phyla reveal several distinguishing features. While the radical SAM domain containing the CX₃CX₂C motif shows the highest conservation (typically 60-70% sequence identity across species), the substrate-binding TRAM domain exhibits considerably more variation (30-45% identity). This pattern suggests that the core catalytic mechanism has remained largely unchanged throughout bacterial evolution, while substrate recognition has adapted to species-specific variations in the S12 protein sequence.

The following table compares key properties of B. bronchiseptica RimO with homologs from selected bacterial species:

Notable structural differences include an extended loop region (residues 320-335) in the B. bronchiseptica RimO that is absent or significantly shorter in most other bacterial homologs. This loop, positioned near the substrate-binding site, may contribute to the specificity for the Bordetella S12 sequence context. Additionally, B. bronchiseptica RimO contains a unique N-terminal extension (residues 1-25) that is predicted to form an amphipathic helix, potentially mediating protein-protein interactions specific to Bordetella species.

Functionally, B. bronchiseptica RimO demonstrates more stringent substrate specificity compared to homologs from some other species. While E. coli RimO can modify synthetic peptides containing the core recognition sequence with relatively high efficiency, B. bronchiseptica RimO requires additional flanking sequences for optimal activity, suggesting a more context-dependent recognition mechanism.

Regulatory differences are also significant, with B. bronchiseptica rimO expression showing distinct responses to environmental signals compared to homologs in other species. In particular, B. bronchiseptica rimO expression is strongly induced under iron-limited conditions, consistent with a Fur box in its promoter region, whereas this regulation is less pronounced in many other bacterial species.

What insights can structural comparisons between B. bronchiseptica RimO and eukaryotic radical SAM enzymes provide?

Structural comparisons between B. bronchiseptica RimO and eukaryotic radical SAM enzymes reveal fascinating evolutionary relationships and highlight unique features that could be exploited for selective targeting. While eukaryotes lack direct homologs of RimO, they possess several radical SAM enzymes involved in diverse cellular processes including vitamin biosynthesis, tRNA modification, and DNA repair. These comparisons provide insights into both conserved catalytic mechanisms and divergent structural adaptations.

Superposition of the predicted structure of B. bronchiseptica RimO with crystal structures of eukaryotic radical SAM enzymes reveals the following key similarities and differences:

One of the most striking differences between B. bronchiseptica RimO and eukaryotic radical SAM enzymes lies in the architecture of the auxiliary cluster binding site. While bacterial RimO enzymes contain a distinct domain that coordinates the auxiliary [4Fe-4S] cluster, eukaryotic radical SAM enzymes that require additional clusters often incorporate them through different structural arrangements or utilize separate protein subunits.

The substrate binding domains also show significant divergence. B. bronchiseptica RimO employs a TRAM domain for S12 recognition, while eukaryotic radical SAM enzymes typically utilize different domain architectures for substrate binding. For instance, human CDKAL1 (which catalyzes the methylthiolation of tRNA) contains a TRAM domain but in a different arrangement relative to the core radical SAM domain, reflecting the different substrate specificities.

These structural comparisons have important implications for drug design. The differences in active site architecture between bacterial RimO and the most closely related eukaryotic enzymes provide opportunities for developing inhibitors with high selectivity for the bacterial enzymes. In particular, the unique arrangement of the auxiliary cluster and substrate binding domains in B. bronchiseptica RimO presents potential binding sites for inhibitors that would not interact with eukaryotic radical SAM enzymes.