Recombinant Prochlorococcus marinus subsp. pastoris (Dimethylallyl)adenosine tRNA methylthiotransferase MiaB (miaB)

What is MiaB and what is its primary function in cellular biology?

MiaB (methylthiotransferase MiaB) is a bifunctional radical-S-adenosylmethionine (radical-AdoMet) enzyme that catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in transfer RNAs (tRNAs) . This modification process involves the insertion of a methyl sulfur group into an unreactive C–H bond, which is a chemically challenging reaction . The methylthiolation of tRNA is crucial for proper translation fidelity and efficiency, as these modifications help stabilize codon-anticodon interactions during protein synthesis. MiaB belongs to the methylthiotransferase (MTTase) family, a subclass of the radical-SAM superfamily that is highly conserved across all three domains of life .

How does the structure of MiaB relate to its catalytic function?

MiaB contains two distinct [4Fe-4S]²⁺'¹⁺ clusters that are essential for its catalytic activity . The first cluster is coordinated by three conserved cysteines (Cys150, Cys154, and Cys157 in Thermotoga maritima MiaB) in the radical-AdoMet motif, while the second cluster is coordinated by three N-terminal conserved cysteines (Cys10, Cys46, and Cys79) . While both clusters have similar UV-visible absorption, resonance Raman, and Mössbauer properties, they differ in their redox properties and EPR characteristics when reduced .

The presence of two iron-sulfur clusters is fundamental to MiaB's catalytic mechanism. The radical-AdoMet cluster generates a 5'-deoxyadenosyl radical that abstracts a hydrogen atom from the substrate, while the auxiliary cluster is believed to function as a sacrificial sulfur donor in the methylthiolation reaction . This structural arrangement enables MiaB to perform the challenging insertion of a methylthio group into an unreactive C-H bond.

What is the evolutionary significance of MiaB and related enzymes?

The MiaB-like protein family, now referred to as the methylthiotransferase (MTTase) family, forms a distinct subclass of the radical-SAM superfamily with high evolutionary conservation . Phylogenetic analysis has revealed that the MTTase family contains only four apparent subfamilies, each with specific functions related to methylthiolation of different substrates .

At least one MiaB-like protein is encoded in most sequenced genomes from all three domains of life, suggesting that this enzyme family originated before the last universal common ancestor . This high degree of conservation underscores the fundamental importance of methylthiolation modifications in cellular processes across diverse life forms. The evolutionary relationship between tRNA-modifying MTTases like MiaB and protein-modifying MTTases like RimO provides fascinating insights into the evolution of substrate specificity in modifying enzymes .

Why is Pichia pastoris chosen as an expression system for recombinant MiaB?

Pichia pastoris has emerged as a highly successful expression system for recombinant proteins due to several key advantages relevant to MiaB production. This yeast expression system provides appropriate protein folding in the endoplasmic reticulum and efficient secretion of recombinant proteins to the external environment through the Kex2 signal peptidase pathway . For complex proteins like MiaB that contain iron-sulfur clusters, proper folding is essential for functional activity.

Additionally, P. pastoris produces limited endogenous secretory proteins, which significantly simplifies the purification process for recombinant MiaB . The system also offers proper post-translational modifications with glycosylation patterns more similar to mammalian cells than bacterial expression systems . For researchers studying MiaB, these features provide considerable advantages over bacterial expression systems where refolding is usually required, while avoiding the high costs and complexity associated with mammalian cell culture systems, as shown in the comparative table below :

What methodological considerations are important when expressing Prochlorococcus marinus MiaB in Pichia pastoris?

When expressing Prochlorococcus marinus MiaB in P. pastoris, several critical methodological considerations must be addressed:

• Codon optimization: The AT-rich genome of Prochlorococcus (GC content of 30.8%) may require codon optimization for efficient expression in P. pastoris, which has a GC content of approximately 38% .

• Expression vector selection: Choosing the appropriate promoter is crucial. For MiaB expression, the methanol-inducible AOX1 promoter is commonly used, but optimization of methanol and sorbitol concentrations is necessary to achieve maximum production .

• Selection of Mut phenotype: Depending on the desired expression profile, researchers must choose between Mut⁺ (methanol utilization positive) or Mut⁻ (methanol utilization negative) strains, which will affect the induction protocol and expression kinetics .

• Iron-sulfur cluster assembly: As MiaB contains two essential [4Fe-4S] clusters , supplementation with iron sources and optimization of growth conditions to support iron-sulfur cluster assembly is critical for obtaining functionally active enzyme.

• Temperature and incubation time optimization: These parameters must be adjusted specifically for MiaB expression, as optimal conditions vary depending on the target protein .

• Purification strategy: Since MiaB contains iron-sulfur clusters, purification must be performed under anaerobic or low-oxygen conditions to prevent cluster degradation, which requires specialized equipment and techniques.

How can the integrity of the recombinant MiaB genome be verified after expression in Pichia pastoris?

Verifying the integrity of recombinant MiaB after expression in P. pastoris is essential due to the potential for mutations during the cloning and expression process. Based on studies with Prochlorococcus genomes cloned in yeast, several methodological approaches are recommended :

• Complete genome sequencing: Comprehensive sequencing of the MiaB gene extracted from P. pastoris is necessary to identify potential mutations. Previous studies with Prochlorococcus genomes identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon, and one dinucleotide transversion compared to the donor genomic DNA .

• Protein mass spectrometry: This technique can verify the primary structure of the expressed protein and detect any variations from the expected sequence.

• Iron and sulfur content analysis: Quantitative analysis of iron and sulfur content using colorimetric assays or ICP-MS can confirm the presence of the expected [4Fe-4S] clusters in the recombinant MiaB.

• Spectroscopic characterization: UV-visible absorption, resonance Raman, and Mössbauer spectroscopy can provide detailed information about the integrity of the iron-sulfur clusters in the recombinant MiaB .

• Activity assays: Functional assays measuring the methylthiolation activity of recombinant MiaB on appropriate tRNA substrates provide the ultimate verification of proper expression and folding.

What techniques are most effective for studying the iron-sulfur clusters in recombinant MiaB?

Detailed characterization of the two essential [4Fe-4S] clusters in MiaB requires a multi-technique approach:

• UV-visible absorption spectroscopy: This technique provides initial confirmation of iron-sulfur cluster incorporation, with characteristic absorption features at approximately 320 and 420 nm for [4Fe-4S]²⁺ clusters .

• Electron Paramagnetic Resonance (EPR) spectroscopy: EPR is crucial for distinguishing between the two [4Fe-4S]¹⁺ clusters in reduced MiaB. The clusters exhibit different EPR properties despite their similar absorption spectra, allowing researchers to monitor both clusters independently .

• Mössbauer spectroscopy: This technique provides detailed information about the oxidation states and electronic environments of the iron atoms in both clusters. It can confirm the presence of [4Fe-4S]²⁺ clusters and monitor changes during the catalytic cycle .

• Resonance Raman spectroscopy: This method provides information about the Fe-S stretching modes in the clusters, helping to characterize their structural integrity and environment .

• Redox titrations: These experiments can determine the redox potentials of the two clusters, which differ despite their similar spectroscopic properties .

• Site-directed mutagenesis: Mutation of the conserved cysteine residues coordinating each cluster can help assign spectroscopic features to specific clusters and understand their individual roles. For example, the C150/154/157A triple variant can be used to study the properties of the N-terminal cluster in isolation .

How does the mechanism of MiaB-catalyzed methylthiolation differ from other radical-SAM enzymes?

MiaB employs a unique dual-function mechanism that distinguishes it from many other radical-SAM enzymes:

• Radical generation: Like all radical-SAM enzymes, MiaB uses its radical-AdoMet [4Fe-4S] cluster to reductively cleave S-adenosylmethionine, generating a 5'-deoxyadenosyl radical. This radical then abstracts a hydrogen atom from the substrate, creating a substrate radical .

• Auxiliary cluster function: Uniquely, MiaB's second [4Fe-4S] cluster serves as a sacrificial sulfur donor in the methylthiolation reaction . This differs from most radical-SAM enzymes that lack a second cluster or use it for other functions.

• Substrate positioning: Recent structural studies have revealed that the methylthio group on MiaB's auxiliary cluster induces a critical change in geometry at the tRNA modification site. This conformational change positions the hydrogen for abstraction by the radical and places the methylthio group in optimal position for subsequent transfer .

• Dual-function catalysis: MiaB performs both methylation and thiolation in a coordinated process, whereas many other radical-SAM enzymes catalyze only a single chemical transformation .

• Cluster regeneration: After each catalytic cycle, the auxiliary cluster must be rebuilt to enable multiple turnovers, a process that is still under investigation and represents a unique aspect of MiaB's catalytic cycle compared to other radical-SAM enzymes .

What is the relationship between MiaB and RimO, and how do their functions differ?

MiaB and RimO are closely related members of the methylthiotransferase (MTTase) family, but they target different substrates with remarkable specificity:

• Substrate specificity: While MiaB catalyzes the methylthiolation of N-6-isopentenyladenosine in tRNAs , RimO methylthiolates the universally conserved aspartate 88 (D88) in ribosomal protein S12 .

• Structural similarity: Despite their different substrates, MiaB and RimO share high structural similarity, both containing two [4Fe-4S] clusters with similar coordination environments and functions in their respective reactions .

• Evolutionary relationship: Phylogenetic analysis places MiaB and RimO in separate subfamilies within the MTTase family, suggesting divergent evolution from a common ancestor to accommodate different substrates .

• Mechanistic parallels: Both enzymes are believed to use similar radical-based mechanisms for methylthiolation, with the second iron-sulfur cluster serving as a sulfur donor in both cases .

• Biological roles: While MiaB's tRNA modifications impact translation efficiency and fidelity broadly, RimO's modification of ribosomal protein S12 appears to have more specialized functions that are still being investigated .

The strong resemblance between these enzymes offers valuable insights into the evolution of substrate specificity in modifying enzymes and provides an excellent comparative system for structural studies .

What are the optimal conditions for in vitro reconstitution of MiaB iron-sulfur clusters?

The successful reconstitution of MiaB's two essential [4Fe-4S] clusters requires careful attention to experimental conditions:

• Anaerobic environment: All reconstitution procedures must be performed in an anaerobic chamber or using Schlenk techniques to prevent oxidative damage to the iron-sulfur clusters .

• Reduction step: Prior to cluster reconstitution, the protein must be reduced with dithiothreitol (DTT) or another suitable reducing agent to ensure the cysteine residues are available for cluster coordination .

• Iron source: Ferrous ammonium sulfate (Fe(NH₄)₂(SO₄)₂) is typically used as the iron source, added in slight excess (8-10 molar equivalents per protein monomer) to ensure complete reconstitution of both clusters .

• Sulfur source: Sodium sulfide (Na₂S) serves as the sulfur source, added in similar excess to the iron source .

• Incubation conditions: The reconstitution mixture should be incubated at 4°C for several hours to overnight, with gentle stirring to promote complete cluster assembly .

• Purification post-reconstitution: After reconstitution, the protein should be purified by size-exclusion chromatography under anaerobic conditions to remove excess iron and sulfide .

• Verification of reconstitution: UV-visible spectroscopy can confirm successful reconstitution, with absorbance at ~420 nm indicating the presence of [4Fe-4S]²⁺ clusters. Iron and sulfide content analyses should yield approximately 8 iron and 8 sulfide atoms per MiaB monomer when both clusters are fully reconstituted .

Reconstituted forms of MiaB containing two [4Fe-4S] clusters have been shown to be significantly more active than partially reconstituted forms, highlighting the importance of optimizing this process .

How can researchers effectively design activity assays for recombinant MiaB?

Designing robust activity assays for recombinant MiaB requires careful consideration of substrate preparation, reaction conditions, and product detection methods:

• Substrate preparation:

  • Prepare tRNA substrates containing N-6-isopentenyladenosine (i⁶A), either by in vitro transcription followed by enzymatic modification or by isolation from appropriate bacterial strains.

  • Ensure substrate purity through HPLC purification and quality control by mass spectrometry.

• Reaction components:

  • Include purified, reconstituted MiaB (containing both [4Fe-4S] clusters)

  • Add S-adenosylmethionine (SAM) as the methyl donor and radical source

  • Include a suitable electron donor system (e.g., sodium dithionite or a flavodoxin/flavodoxin reductase/NADPH system)

  • Buffer composition should maintain anaerobic conditions and appropriate pH (typically 7.0-8.0)

• Reaction conditions:

  • Perform assays under strictly anaerobic conditions to maintain cluster integrity

  • Incubate at physiologically relevant temperatures (typically 30-37°C)

  • Monitor time-dependent product formation to establish linear reaction rates

• Product detection methods:

  • HPLC analysis of nucleosides after enzymatic digestion of tRNA

  • Mass spectrometry to confirm methylthiolation by the characteristic mass increase

  • Radiometric assays using ¹⁴C-labeled SAM to track methyl group transfer

• Controls:

  • Negative controls should include reactions without enzyme, without SAM, or with heat-inactivated enzyme

  • Positive controls could include previously characterized bacterial MiaB enzymes

  • Use variant forms of MiaB with mutations in either Fe-S cluster to distinguish the roles of each cluster

What bioinformatic resources are valuable for studying MiaB and related methylthiotransferases?

Several bioinformatic resources provide valuable information for researchers studying MiaB and related methylthiotransferases:

• IntEnzyDB: This integrated structure-kinetics enzymology database employs a relational database architecture with a flattened data structure, allowing researchers to map enzyme structure and function data. IntEnzyDB contains enzyme kinetics and structure data from six enzyme commission classes and can help researchers investigate the effects of mutations on enzyme activity .

• Structure prediction tools: For MiaB or related enzymes without solved crystal structures, tools like AlphaFold2 can generate accurate structure predictions based on sequence information and evolutionary relationships.

• Sequence alignment and phylogenetic analysis tools: These are essential for classifying MiaB variants and related enzymes. Previous studies have identified four apparent subfamilies within the methylthiotransferase family through such analyses .

• Databases of tRNA modifications: Resources like the Modomics database (http://modomics.genesilico.pl/) provide information on known tRNA modifications, including those catalyzed by MiaB, and can help identify potential substrates and products.

• Protein-protein interaction databases: These resources can help identify potential interaction partners of MiaB, which may be involved in cluster assembly, electron transfer, or substrate recognition.

• Gene co-expression databases: These can provide insights into the cellular processes in which MiaB participates and may suggest functional relationships with other genes.

• Comparative genomics tools: These allow researchers to study the distribution and conservation of MiaB across different species and could reveal evolutionary patterns and functional adaptations.

How does the auxiliary [4Fe-4S] cluster in MiaB get rebuilt after each catalytic cycle?

The mechanism by which the auxiliary [4Fe-4S] cluster in MiaB is rebuilt after donating its sulfur atom during catalysis remains a major research question . Several hypothetical pathways have been proposed:

• Iron-sulfur cluster biogenesis machinery involvement: Cellular iron-sulfur cluster assembly systems (ISC, SUF, or NIF) may directly interact with MiaB to repair or rebuild its auxiliary cluster after each turnover.

• Small molecule sulfur donors: Cellular small molecule sulfur carriers like cysteine, glutathione, or persulfides might donate sulfur atoms to rebuild the degraded cluster.

• Specialized repair proteins: Dedicated repair proteins may exist that specifically recognize and repair damaged auxiliary clusters in MiaB and related enzymes.

• Self-repair mechanism: MiaB might contain additional domains or features that facilitate self-repair of the auxiliary cluster using cellular sulfur sources.

• Multiple enzyme forms: Cells might maintain a pool of fully reconstituted MiaB enzymes, with "spent" enzymes being replaced rather than repaired for each catalytic cycle.

Investigating this question requires advanced techniques such as time-resolved spectroscopy to capture intermediate states during cluster rebuilding, protein-protein interaction studies to identify potential repair partners, and in vivo studies using isotopically labeled sulfur sources to track the origin of replacement sulfur atoms .

What are the structural determinants of substrate specificity between MiaB and other methylthiotransferases like RimO?

The remarkable substrate specificity exhibited by MiaB (targeting tRNA) and RimO (targeting ribosomal protein S12) despite their structural similarity raises fascinating questions about the determinants of this specificity :

• Substrate binding domains: Comparative structural analysis of MiaB and RimO may reveal distinct substrate binding pockets or domains that recognize either tRNA or protein substrates. These could involve differences in surface charge distribution, hydrophobicity patterns, or specific recognition motifs.

• Conformational dynamics: The two enzymes might share similar active sites but exhibit different conformational dynamics that accommodate their respective substrates. Advanced techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) could reveal these differences.

• Accessory proteins: In vivo, MiaB and RimO might interact with different accessory proteins that help position their specific substrates correctly. Protein-protein interaction studies could identify such partners.

• Recognition sequence context: Analysis of the sequences surrounding the modification sites in both substrates might reveal specific recognition patterns that direct enzyme specificity.

• Evolutionary analysis: Detailed phylogenetic analysis coupled with ancestral sequence reconstruction could reveal the evolutionary path by which these enzymes diverged to acquire different specificities .

Understanding these determinants could enable the rational design of MTTases with novel substrate specificities for biotechnological applications or provide insights into the evolution of enzyme specificity more broadly.

How might the structure and function of MiaB be affected by incorporation into multi-enzyme complexes?

The potential incorporation of MiaB into multi-enzyme complexes represents an unexplored frontier with significant implications:

• tRNA modification factories: MiaB might participate in organized "modification factories" where multiple tRNA-modifying enzymes work in concert. This arrangement could enhance efficiency through substrate channeling, where modified tRNA is passed directly from one enzyme to the next.

• Cluster assembly interactions: MiaB likely interacts with the iron-sulfur cluster biogenesis machinery, particularly after catalysis when its auxiliary cluster needs rebuilding. These interactions might involve transient or stable complexes that facilitate efficient cluster repair.

• Electron transfer partnerships: As a radical-SAM enzyme, MiaB requires electrons to initiate its reaction. It might form complexes with electron transfer proteins like flavodoxins or ferredoxins that provide the necessary reducing equivalents.

• Regulatory interactions: MiaB activity might be regulated through interactions with sensor proteins that respond to cellular conditions like nutrient availability or oxidative stress, coordinating tRNA modification with cellular needs.

• Membrane associations: In some systems, tRNA modification enzymes are associated with membranes. If MiaB forms such associations, its structure and function could be significantly affected by the membrane environment.

Investigating these potential interactions requires techniques like cryo-electron microscopy, native mass spectrometry, in-cell fluorescence microscopy, and cross-linking studies coupled with proteomics to identify and characterize potential MiaB-containing complexes.