Recombinant Neosartorya fumigata AdoMet-dependent rRNA methyltransferase spb1 (spb1), partial

What is the SPB1 methyltransferase in Neosartorya fumigata and what is its function?

SPB1 from Neosartorya fumigata (strain ATCC MYA-4609/Af293/CBS 101355/FGSC A1100) is an AdoMet-dependent rRNA methyltransferase that catalyzes the transfer of methyl groups from S-adenosyl methionine (AdoMet) to specific positions on ribosomal RNA . Similar to other rRNA methyltransferases such as RlmM in E. coli, SPB1 likely plays a role in ribosome biogenesis and function through post-transcriptional modification of rRNA . These modifications typically occur during ribosome assembly and can influence ribosomal structure, function, and antibiotic susceptibility. In particular, 2′O methylation of ribose moieties in rRNA can stabilize RNA structure through altered hydrogen bonding and stacking interactions.

How does SPB1 compare structurally to other known methyltransferases?

Based on comparative analysis with similar methyltransferases like RlmM from E. coli, SPB1 likely contains a catalytic Rossmann-like fold MTase domain characteristic of AdoMet-dependent methyltransferases . Many rRNA methyltransferases contain a conserved K-D-K-E/H catalytic tetrad necessary for methyl transfer from AdoMet to the target rRNA site . SPB1 may also contain an RNA-binding domain, potentially similar to the N-terminal THUMP domain found in RlmM, which assists in substrate recognition . Notably, in similar enzymes like RlmM, the AdoMet-binding site is often relatively open and shallow, suggesting that RNA substrate binding may be required to induce conformational changes necessary for optimal catalytic activity .

What is the substrate specificity of SPB1 methyltransferase?

While specific experimental data on SPB1 from N. fumigata is limited in the provided search results, inferences can be made from similar enzymes. SPB1 likely shows specificity for particular nucleotides within ribosomal RNA, similar to how RlmM targets C2498 in 23S rRNA of E. coli . The enzyme probably recognizes structural features of the rRNA target site rather than simple sequence motifs. Studies of RlmM have shown that it can modify in vitro transcribed 23S rRNA and even domain V alone, suggesting that recognition doesn't necessarily require other rRNA domains or prior modifications . For SPB1, determining whether it acts on naked rRNA or partially assembled ribonucleoprotein particles would provide important insights into its role in ribosome biogenesis.

What expression systems are optimal for producing recombinant N. fumigata SPB1?

For recombinant expression of N. fumigata proteins, E. coli expression systems have been successfully employed as evidenced by the production of other N. fumigata proteins like RODA . When designing an expression construct for SPB1, consider including an affinity tag (such as a 6xHis tag) to facilitate purification . Since SPB1 is likely involved in RNA interactions, ensuring proper folding is critical. Therefore, expression conditions should be optimized through testing different E. coli strains (BL21(DE3), Rosetta, Arctic Express), induction temperatures (16-37°C), and IPTG concentrations (0.1-1.0 mM). Alternative expression systems such as yeast (P. pastoris) might be considered if E. coli expression yields insoluble protein. Consider co-expression with molecular chaperones if misfolding occurs, and evaluate the effect of tags on enzymatic activity.

How can the methyltransferase activity of SPB1 be assayed in vitro?

The methyltransferase activity of SPB1 can be assayed using methods similar to those employed for other rRNA methyltransferases. One effective approach is the primer extension assay under limiting dNTP concentrations, which can detect 2′O methylation as it causes reverse transcriptase to pause one nucleotide before the methylation site . For this assay:

• Incubate purified recombinant SPB1 with in vitro transcribed rRNA substrate and AdoMet.

• Extract and purify the RNA.

• Perform primer extension using reverse transcriptase under limiting dNTP conditions.

• Analyze the extension products by gel electrophoresis to identify methylation-dependent stops.

Alternative methods include:

• Thin-layer chromatography (TLC) with radiolabeled AdoMet to track methyl transfer

• Mass spectrometry to detect the mass increase from methylation

• AdoMet-to-AdoHcy conversion assays using coupled enzyme systems

What purification strategy is recommended for recombinant SPB1?

Based on strategies used for similar proteins, a multi-step purification protocol for His-tagged recombinant SPB1 would include:

Important considerations include maintaining protein stability through the addition of glycerol (10-20%) to storage buffers and avoiding repeated freeze-thaw cycles . For long-term storage, aliquot the purified protein and store at -80°C, potentially with 50% glycerol as a cryoprotectant . Verify the purity using SDS-PAGE (aiming for >85% purity) and confirm activity through functional assays before proceeding with structural or biochemical studies .

How does substrate binding influence the catalytic activity of SPB1?

Based on studies of similar methyltransferases like RlmM, SPB1 likely undergoes conformational changes upon substrate binding that are essential for optimal catalytic activity . In RlmM, the AdoMet-binding site is relatively open and shallow in the absence of RNA substrate, suggesting that substrate binding may induce structural changes necessary for proper positioning of the methyl donor .

This substrate-induced activation mechanism has been observed in other RNA methyltransferases:

• Protein-mediated stabilization: In some systems, auxiliary proteins stabilize the motif regions around the AdoMet binding site, increasing affinity for AdoMet .

• RNA-mediated stabilization: For methyltransferases like KsgA, substrate binding triggers conformational changes that stabilize motifs essential for efficient AdoMet binding .

For SPB1, in vitro activity assays comparing the enzyme's affinity for AdoMet in the presence and absence of RNA substrate could elucidate whether it employs a similar mechanism. Structural studies (X-ray crystallography or cryo-EM) of SPB1 alone and in complex with RNA substrate would provide definitive evidence for such conformational changes.

What is the role of SPB1 in ribosome assembly and antibiotic resistance in N. fumigata?

While direct evidence for SPB1's role in N. fumigata is limited in the provided search results, insights can be drawn from related methyltransferases. Similar to how RlmM acts during early stages of ribosome assembly in E. coli , SPB1 likely functions during specific stages of ribosome biogenesis in N. fumigata. In E. coli, RlmM is active on protein-free 23S rRNA but not on mature 50S subunits, indicating it acts early in assembly . The modification site in mature ribosomes is often inaccessible, supporting this temporal requirement .

Regarding antibiotic resistance, rRNA modifications can influence antibiotic susceptibility by altering the binding sites for antibiotics that target the ribosome. For N. fumigata, a clinically important fungal pathogen, SPB1-mediated modifications could potentially contribute to intrinsic or acquired resistance to antifungal agents. Knockout studies of SPB1 in N. fumigata, similar to those conducted for RlmM in E. coli (which showed reduced fitness in competition assays ), would be valuable for assessing its physiological importance.

How do the structural features of SPB1 compare across different Aspergillus species?

Comparative analysis of SPB1 across Aspergillus and related fungal species would reveal conserved structural elements essential for function versus species-specific adaptations. Based on patterns observed in other methyltransferases, we would expect:

• Catalytic domain conservation: The Rossmann-like fold MTase domain and catalytic K-D-K-E/H tetrad are likely highly conserved across species due to their essential role in methyl transfer .

• RNA-binding domain variation: The RNA-binding domains may show greater sequence divergence while maintaining structural similarity, potentially reflecting adaptations to species-specific rRNA structures.

• Species-specific insertions or extensions: These might modulate substrate specificity or interactions with species-specific ribosomal assembly factors.

Phylogenetic analysis combined with homology modeling based on available crystal structures of related methyltransferases would provide insights into evolutionary relationships and structural conservation. Such comparative analysis could identify conserved surface patches likely involved in substrate recognition versus variable regions that might contribute to species-specific functions.

How can researchers differentiate between enzymatic and non-enzymatic methylation in SPB1 activity assays?

Distinguishing enzymatic from non-enzymatic methylation is critical for accurately assessing SPB1 activity. Several experimental controls and analytical approaches can address this challenge:

• Negative controls: Include reactions without enzyme, without AdoMet, and with heat-inactivated enzyme to establish baseline non-enzymatic methylation levels.

• Catalytic mutants: Generate site-directed mutants of predicted catalytic residues (particularly in the K-D-K-E/H tetrad) to create catalytically inactive versions of SPB1 that should still bind RNA but not catalyze methyl transfer .

• Time-course analysis: Enzymatic reactions show characteristic time-dependent kinetics that differ from the typically linear progression of non-enzymatic reactions.

• Substrate specificity: Test methylation of non-specific RNA substrates versus the natural substrate; enzymatic methylation should show significantly higher specificity.

• Response to environmental conditions: Enzymatic reactions typically show bell-shaped pH profiles and temperature dependence characteristic of protein catalysts.

For primer extension assays specifically, compare band intensities at the methylation site relative to control stops (as done in RlmM studies ) to quantify methylation levels.

What computational approaches are useful for predicting SPB1 substrate recognition sites?

Predicting SPB1 substrate recognition sites requires integrative computational approaches:

• Sequence conservation analysis: Align rRNA sequences from related fungal species to identify conserved regions that might contain methylation sites.

• RNA secondary structure prediction: Tools like RNAfold, Mfold, or RNAstructure can predict the secondary structure context of potential target sites.

• Motif identification: Machine learning algorithms trained on known methyltransferase target sites can identify sequence or structural motifs associated with 2′O methylation.

• Molecular docking: Using homology models of SPB1 based on related methyltransferases like RlmM , dock potential RNA substrates to predict binding modes and energetics.

• Molecular dynamics simulations: These can reveal dynamic interactions between SPB1 and RNA substrates, particularly conformational changes induced by binding.

The results from these computational approaches should be validated experimentally, potentially using methods like site-directed mutagenesis of predicted target nucleotides followed by in vitro methylation assays.

How should researchers interpret discrepancies between in vitro and in vivo methylation patterns of SPB1?

Discrepancies between in vitro and in vivo methylation patterns are common in rRNA methyltransferase research and can provide valuable insights into biological context:

• Ribosome assembly factors: In vivo, ribosomal proteins and assembly factors may restrict access to certain rRNA regions or enhance specificity for others. Studies with RlmM showed it can act on naked 23S rRNA in vitro but in vivo acts on rRNA with some ribosomal proteins already bound .

• Temporal regulation: The timing of SPB1 activity during ribosome assembly may differ between in vitro and in vivo conditions. In E. coli, RlmM acts early in 50S assembly when the modification site is accessible, but not on mature 50S subunits .

• Cooperative modifications: Other rRNA modifications may influence SPB1 activity through structural changes that enhance or inhibit substrate recognition.

• Cellular compartmentalization: In vivo, spatial organization within the nucleus or nucleolus may concentrate enzymes and substrates, affecting reaction efficiency.

To address these discrepancies, researchers should consider using partially reconstituted in vitro systems that better mimic in vivo conditions, such as including relevant ribosomal proteins or performing activity assays on ribosome assembly intermediates isolated from cells.

What emerging technologies could advance our understanding of SPB1 function?

Several cutting-edge technologies offer promising approaches for elucidating SPB1 function:

• Cryo-electron microscopy (cryo-EM): This technique could capture different conformational states of SPB1 during catalysis and visualize its interaction with rRNA substrates at near-atomic resolution.

• Single-molecule fluorescence resonance energy transfer (smFRET): This approach could monitor real-time conformational changes in SPB1 upon substrate binding and during catalysis.

• Nanopore sequencing: Direct RNA sequencing using nanopore technology can detect rRNA modifications without conversion to cDNA, potentially revealing the complete modification landscape influenced by SPB1.

• CRISPR-Cas9 base editing: This technique allows for precise modification of specific nucleotides in rRNA genes to study the effects of altering SPB1 target sites.

• Time-resolved X-ray crystallography: This could capture transient catalytic intermediates during the methyl transfer reaction.

These technologies, combined with traditional biochemical and genetic approaches, would provide a comprehensive understanding of SPB1 function in ribosome biogenesis and potentially uncover novel roles in fungal physiology and pathogenesis.

How might SPB1 function differ between laboratory and clinical isolates of N. fumigata?

Laboratory strains like ATCC MYA-4609/Af293/CBS 101355/FGSC A1100 and clinical isolates of N. fumigata may exhibit differences in SPB1 function due to selective pressures in their respective environments:

• Expression levels: Clinical isolates may show altered expression of SPB1 in response to host environments or antifungal treatments.

• Sequence variations: Natural polymorphisms in SPB1 could affect substrate specificity, catalytic efficiency, or interactions with ribosome assembly factors.

• Modification patterns: The specific rRNA sites modified by SPB1 might differ between strains, potentially influencing ribosome function and antibiotic susceptibility.

• Regulatory differences: The regulation of SPB1 expression or activity in response to stress conditions relevant to infection (temperature, pH, oxidative stress) may vary between laboratory and clinical isolates.

Comparative genomic, transcriptomic, and biochemical analyses of SPB1 from laboratory and clinical isolates would reveal potential adaptations relevant to pathogenesis. Additionally, determining whether SPB1 contributes to antifungal resistance in clinical isolates could have significant implications for therapeutic strategies against N. fumigata infections.