Recombinant Exiguobacterium sibiricum tRNA dimethylallyltransferase (miaA)

What is Exiguobacterium sibiricum and why is it significant for MiaA research?

Exiguobacterium sibiricum is a gram-positive, non-spore-forming, rod-shaped bacterium belonging to the genus Exiguobacterium. This organism is particularly significant for enzyme research due to its psychrotolerant properties, being capable of growth at temperatures as low as 4°C . As a member of the coryneform bacteria group, E. sibiricum exhibits several distinctive characteristics: it grows aerobically, is motile, catalase-positive, and oxidase-negative . The bacterium forms mucoid, initially gray colonies that turn orange after 48 hours of incubation on blood agar, with no hemolysis observed .

E. sibiricum is an ideal source for MiaA research because its adaptation to extreme environments may confer unique structural and functional properties to its enzymes. The bacterium's ability to thrive in cold conditions suggests that its enzymes, including MiaA, may possess specialized adaptations for functionality at lower temperatures, making them valuable models for understanding enzyme cold adaptation mechanisms.

What is tRNA dimethylallyltransferase (MiaA) and what function does it serve?

tRNA dimethylallyltransferase, encoded by the miaA gene, is an enzyme that catalyzes a critical step in tRNA modification. Specifically, it transfers a five-carbon isoprenoid moiety from dimethylallyl pyrophosphate (DMAPP) to the amino group of adenosine at position 37 of specific tRNAs . This modification, located adjacent to the anticodon, enhances codon-anticodon interactions during translation, thereby improving translational efficiency and fidelity.

Structurally, MiaA (also called DMATase) possesses a central channel spanning the entire width of the enzyme. This architecture accommodates both substrates—tRNA enters from one side and DMAPP from the opposite side—in an ordered sequence, with tRNA binding first followed by DMAPP . The modification reaction occurs in the middle of this channel where the two substrates meet. Interestingly, MiaA's structure shows homology to a class of small soluble kinases involved in nucleotide precursor biosynthesis, suggesting its evolutionary origin .

What growth conditions are optimal for culturing Exiguobacterium sibiricum?

For effective cultivation of E. sibiricum, researchers should consider the following growth parameters:

• Temperature range: The organism can grow at temperatures as low as 4°C (demonstrated by growth on blood agar after 6 days of incubation) . Standard laboratory cultivation typically occurs at 30-37°C for faster growth.

• Media: Blood agar supports growth well, with distinctive colony morphology (gray colonies turning orange after 48 hours) .

• Oxygen requirements: E. sibiricum is facultatively anaerobic, meaning it can grow in both aerobic and anaerobic conditions .

• Growth characteristics: Colonies appear mucoid and non-hemolytic on blood agar .

The ability to grow at 4°C is a distinguishing characteristic that differentiates E. sibiricum from related bacteria like Bacillus anthracis, which cannot grow at this temperature . This psychrotolerant nature is particularly important for researchers interested in cold-adapted enzymes.

How is E. sibiricum identified and differentiated from similar organisms?

Accurate identification of E. sibiricum requires a combination of traditional microbiological methods and molecular techniques. The table below outlines key differentiating characteristics between E. sibiricum and B. anthracis, which can appear similar in some clinical presentations:

Conventional identification methods can be difficult and should be confirmed with molecular assays . Definitive identification requires 16S rRNA gene sequencing, which has been shown to reliably identify E. sibiricum with high accuracy (99.6% similarity to reference sequences) .

What are the structural characteristics of MiaA from E. sibiricum compared to orthologs from other organisms?

The structure of tRNA dimethylallyltransferase reveals several distinctive features that influence its function. Crystal structures of DMATase (at 1.9 Å resolution) show a central channel spanning the width of the enzyme . This architectural feature is critical for the enzyme's function, as it allows:

• Entry of tRNA substrate from one side

• Entry of DMAPP (dimethylallyl pyrophosphate) from the opposite side

• Sequential binding in an ordered manner (tRNA first, DMAPP second)

• Catalysis of the modification reaction at the meeting point in the middle of the channel

While the structure described in the search results is from Pseudomonas aeruginosa DMATase, the E. sibiricum enzyme would likely share many conserved features given the functional constraints of the reaction it catalyzes. The enzyme belongs to a structural family related to small soluble kinases involved in nucleotide precursor biosynthesis, suggesting its evolutionary trajectory .

A distinctive feature of DMATase is its conserved loop for pyrophosphate recognition, similar to the P-loop found in diverse nucleotide-binding proteins . This structural arrangement makes DMATase mechanistically and structurally distinct from farnesyltransferase, another prenyltransferase family involved in protein modification .

What expression systems are most effective for producing recombinant E. sibiricum MiaA?

For optimal expression of recombinant E. sibiricum MiaA, researchers should consider expression systems that accommodate the enzyme's potential cold-adaptation features. Based on general principles for expressing enzymes from psychrotolerant organisms, the following approach is recommended:

• Host selection: Escherichia coli BL21(DE3) strains are commonly used for initial expression trials. For difficult-to-express proteins from cold-adapted organisms, specialized strains like Arctic Express (containing cold-adapted chaperonins) may improve soluble expression.

• Expression vectors: Vectors containing strong inducible promoters (T7, tac) with temperature-modulated induction systems are preferable. Including solubility-enhancing fusion tags such as MBP (maltose-binding protein), SUMO, or thioredoxin can improve soluble expression.

• Induction conditions: Lower induction temperatures (15-20°C) often improve the solubility of recombinant proteins from psychrotolerant organisms by slowing protein synthesis and allowing proper folding. Extended expression periods (overnight) at these reduced temperatures may be necessary.

• Media optimization: Enriched media formulations (such as Terrific Broth) supplemented with osmolytes or compatible solutes may enhance protein stability during expression.

The effectiveness of these strategies may vary, necessitating optimization for the specific recombinant E. sibiricum MiaA construct. Monitoring expression through small-scale trials with different conditions is advised before proceeding to large-scale production.

How can the enzymatic activity of recombinant E. sibiricum MiaA be measured accurately?

Accurate measurement of MiaA activity requires assays that detect the transfer of the dimethylallyl group from DMAPP to the adenosine at position 37 of tRNA. Several methodological approaches can be employed:

• Radioisotope-based assays: Using [14C]- or [3H]-labeled DMAPP as substrate and monitoring the incorporation of radioactivity into tRNA. This approach offers high sensitivity but requires handling of radioactive materials.

• HPLC-based assays: Analyzing the modification status of tRNA substrates before and after incubation with the enzyme. Modified nucleosides exhibit different retention times compared to unmodified ones.

• Mass spectrometry: Detection of the mass shift in tRNA or its digestion products resulting from the addition of the dimethylallyl group. This approach offers high sensitivity and specificity.

• Coupled enzyme assays: Detecting the release of pyrophosphate through coupling with pyrophosphatase and other enzymes that generate a colorimetric or fluorescent signal.

A typical reaction mixture would contain:

• Purified recombinant E. sibiricum MiaA

• Appropriate tRNA substrate (either total tRNA or specific tRNA species)

• DMAPP (dimethylallyl pyrophosphate)

• Buffer system (typically at pH 7.5-8.0)

• Divalent cations (Mg2+ is often required)

• Reducing agent (DTT or β-mercaptoethanol)

For evaluating cold-adaptation properties, activity measurements should be conducted across a temperature range (e.g., 4-37°C) to determine the temperature optimum and activity profile of the enzyme.

What substrate specificities are expected for E. sibiricum MiaA?

Based on knowledge of tRNA dimethylallyltransferases, E. sibiricum MiaA likely exhibits the following substrate specificities:

• tRNA specificity: MiaA typically modifies tRNAs that read codons beginning with U, particularly tRNAPhe, tRNATyr, and tRNATrp. The enzyme recognizes specific structural features in these tRNAs, with the anticodon loop structure being particularly important for substrate recognition.

• Nucleoside specificity: The enzyme specifically modifies adenosine at position 37, which is adjacent to the anticodon. This position is highly conserved in tRNAs that serve as MiaA substrates.

• Prenyl donor specificity: DMAPP (dimethylallyl pyrophosphate) serves as the donor of the five-carbon isoprenoid moiety . The enzyme likely has a specific binding pocket for this substrate, with recognition of the pyrophosphate portion via a conserved loop similar to the P-loop in nucleotide-binding proteins .

• Catalytic mechanism: The reaction appears to proceed through an ordered binding mechanism, with tRNA binding first followed by DMAPP . This order is facilitated by the central channel architecture of the enzyme.

To experimentally determine the substrate specificity of E. sibiricum MiaA, researchers could employ:

• In vitro modification assays with various tRNA species

• Structure-based mutational analysis of the enzyme

• Competition assays with different potential prenyl donors

• Binding studies using isothermal titration calorimetry or surface plasmon resonance

What purification strategies yield highly active recombinant E. sibiricum MiaA?

For optimal purification of recombinant E. sibiricum MiaA while maintaining enzymatic activity, the following multi-step strategy is recommended:

• Cell lysis conditions:

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

  • Addition of protease inhibitors (PMSF or commercial cocktail)

  • Gentle lysis methods (sonication with cooling periods or enzymatic lysis) to prevent protein denaturation

• Initial capture:

  • Affinity chromatography using a fusion tag (His6, MBP, or GST tag)

  • For His-tagged constructs: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Washing with increasing imidazole concentrations (10-40 mM) to remove non-specifically bound proteins

  • Elution with higher imidazole concentration (250-300 mM)

• Intermediate purification:

  • Ion exchange chromatography based on the theoretical pI of the protein

  • For proteins with pI < 7: Q-Sepharose (anion exchange)

  • For proteins with pI > 7: SP-Sepharose (cation exchange)

  • Salt gradient elution (typically 0-1 M NaCl)

• Polishing step:

  • Size exclusion chromatography to remove aggregates and achieve high purity

  • Recommended column: Superdex 200 or Sephacryl S-200

  • Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Special considerations for cold-adapted enzymes:

  • Maintain lower temperatures (4-15°C) throughout purification

  • Include stabilizing agents (glycerol, trehalose, or specific ions)

  • Avoid freeze-thaw cycles that may lead to activity loss

  • Consider rapid purification protocols to minimize time for potential denaturation

The purified enzyme should be assessed for homogeneity using SDS-PAGE and for activity using appropriate enzymatic assays. For long-term storage, flash freezing in liquid nitrogen with cryoprotectants (e.g., 20% glycerol) and storage at -80°C is recommended.

How can crystallization trials for structural studies of E. sibiricum MiaA be optimized?

Optimizing crystallization trials for structural studies of E. sibiricum MiaA requires careful attention to several factors:

• Protein preparation:

  • Ensure high purity (>95% as assessed by SDS-PAGE and size exclusion chromatography)

  • Verify protein homogeneity using dynamic light scattering

  • Determine optimal buffer conditions for stability using thermal shift assays

  • Consider protein engineering approaches (surface entropy reduction, removal of flexible regions) if initial trials fail

• Initial screening:

  • Commercial sparse matrix screens (Hampton Research, Molecular Dimensions, Qiagen)

  • Grid screens based on successful conditions for related enzymes

  • Inclusion of substrate analogs or products to stabilize active site conformations

  • Variation of protein concentration (typically 5-15 mg/ml)

• Optimization strategies:

  • Fine-tuning of precipitant concentration

  • pH optimization (typically in 0.2 unit increments)

  • Additive screening (small molecules, ions, detergents)

  • Seeding techniques (microseed matrix screening)

• Cold-adaptation considerations:

  • Trial crystallization at different temperatures (4°C, 10°C, 18°C)

  • Include cryoprotectants that maintain cold-adapted enzyme conformations

  • Consider co-crystallization with tRNA substrate or DMAPP

• Data collection strategies:

  • Cryoprotection protocols to prevent ice formation

  • In-house preliminary diffraction testing before synchrotron data collection

  • Multiple anomalous dispersion (MAD) or molecular replacement using related structures as search models

Based on information about DMATase structure , considering crystallization with components that stabilize the central channel architecture may be beneficial. Additionally, co-crystallization with pyrophosphate has been successful for related enzymes and could be attempted for E. sibiricum MiaA .

What approaches can be used to investigate the temperature dependence of E. sibiricum MiaA activity?

To comprehensively characterize the temperature dependence of E. sibiricum MiaA activity, researchers should implement the following experimental approaches:

• Temperature-activity profiling:

  • Measure enzyme activity across a wide temperature range (0-50°C)

  • Use appropriate temperature controls and equilibration periods

  • Plot relative activity versus temperature to determine temperature optimum

  • Compare with MiaA enzymes from mesophilic organisms to identify cold-adaptation features

• Thermal stability assessment:

  • Differential scanning calorimetry (DSC) to determine melting temperature (Tm)

  • Circular dichroism (CD) spectroscopy at various temperatures to monitor secondary structure changes

  • Fluorescence-based thermal shift assays for high-throughput screening of stabilizing conditions

  • Activity retention after incubation at various temperatures (thermal inactivation kinetics)

• Kinetic parameter determination at different temperatures:

  • Measure Km and kcat values for both tRNA and DMAPP substrates at multiple temperatures

  • Calculate activation energy (Ea) using Arrhenius plots

  • Determine thermodynamic parameters (ΔH‡, ΔG‡, ΔS‡) using transition state theory

• Structural analysis of temperature effects:

  • Hydrogen-deuterium exchange mass spectrometry at different temperatures

  • Solution nuclear magnetic resonance (NMR) spectroscopy to detect temperature-dependent conformational changes

  • Molecular dynamics simulations to identify regions with temperature-dependent flexibility

• Comparative analysis with homologous enzymes:

  • Side-by-side comparison with MiaA from mesophilic and thermophilic organisms

  • Identification of specific amino acid substitutions associated with cold adaptation

  • Site-directed mutagenesis to verify the role of specific residues in cold adaptation

These approaches collectively provide a comprehensive understanding of how E. sibiricum MiaA has adapted to function at lower temperatures, potentially revealing unique structural and functional features that could be applied to enzyme engineering for low-temperature applications.

What strategies can address poor expression yields of recombinant E. sibiricum MiaA?

Poor expression yields of recombinant E. sibiricum MiaA can be addressed through several methodological interventions:

• Codon optimization strategies:

  • Analyze the codon usage of the native gene compared to the expression host

  • Synthesize a codon-optimized version for expression in E. coli or other expression hosts

  • Consider the use of strains containing rare codon tRNAs (e.g., Rosetta strains)

• Expression construct optimization:

  • Test multiple fusion tags (His, GST, MBP, SUMO, Thioredoxin) to improve solubility

  • Optimize the position of the tag (N-terminal vs. C-terminal)

  • Include TEV or other protease cleavage sites for tag removal

  • Consider domain truncation if the full-length protein expresses poorly

• Expression condition modifications:

  • Reduce induction temperature (15-20°C) to slow protein synthesis and improve folding

  • Test various inducer concentrations (0.01-1 mM IPTG for T7-based systems)

  • Extend expression time at lower temperatures (24-48 hours)

  • Try auto-induction media for gradual protein expression

• Host strain selection:

  • Test expression in multiple E. coli strains (BL21(DE3), C41/C43, Arctic Express)

  • Consider non-E. coli expression systems (Pichia pastoris, insect cells) for difficult proteins

  • Use strains with enhanced disulfide bond formation capabilities if applicable

• Co-expression approaches:

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J) to improve folding

  • Co-express with partners or subunits if the protein functions in a complex

  • Include sigma factors or transcription factors that might regulate expression in the native organism

Systematic testing of these variables through small-scale expression trials, followed by SDS-PAGE and western blot analysis, can identify optimal conditions for subsequent scale-up. For psychrotolerant enzymes like E. sibiricum MiaA, cold-induction strategies may be particularly effective in obtaining correctly folded, active protein.

How can researchers troubleshoot enzymatic assays when E. sibiricum MiaA shows low activity?

When E. sibiricum MiaA exhibits unexpectedly low enzymatic activity, researchers should systematically investigate the following potential issues:

• Protein quality factors:

  • Verify protein integrity using SDS-PAGE and mass spectrometry

  • Check for proteolytic degradation with western blotting

  • Assess protein folding using circular dichroism or fluorescence spectroscopy

  • Determine aggregation state using size exclusion chromatography or dynamic light scattering

• Substrate-related considerations:

  • Confirm tRNA substrate quality (integrity, correct folding)

  • Verify DMAPP purity and stability

  • Test multiple tRNA species as the enzyme may have specific substrate preferences

  • Consider tRNA pre-folding steps or renaturation protocols

• Buffer optimization:

  • Screen different buffer systems (HEPES, Tris, phosphate) across pH range 6.5-8.5

  • Test various salt concentrations (50-300 mM NaCl or KCl)

  • Optimize divalent cation concentration (Mg2+, Mn2+) as they are often essential for activity

  • Include stabilizing agents (glycerol, BSA) to prevent surface denaturation

• Temperature considerations:

  • As a psychrotolerant organism enzyme, E. sibiricum MiaA may have optimal activity at lower temperatures

  • Perform activity assays across a temperature range (4-37°C)

  • Allow sufficient incubation time at lower temperatures to compensate for slower reaction rates

• Detection method refinement:

  • Increase sensitivity of detection methods

  • Extend reaction times for challenging substrates or conditions

  • Consider alternative assay formats if conventional methods yield poor results

  • Use positive controls (MiaA from E. coli or other well-characterized organisms)

A methodical approach to troubleshooting, with careful documentation of all variables changed and their effects on enzyme activity, will help identify the specific factors limiting activity in the experimental system. For cold-adapted enzymes, special attention to temperature-dependent activity profiles is particularly important.

How is research on E. sibiricum MiaA contributing to understanding of tRNA modification in extremophiles?

Research on E. sibiricum MiaA provides valuable insights into tRNA modification mechanisms in extremophile organisms, contributing to several key areas:

• Cold adaptation mechanisms of RNA-modifying enzymes:

  • Analysis of E. sibiricum MiaA can reveal structural and kinetic adaptations that permit enzyme activity at low temperatures

  • Comparison with mesophilic and thermophilic MiaA homologs helps identify specific amino acid substitutions or structural features that confer cold activity

  • These insights extend our understanding of how essential cellular processes like translation adapt to extreme conditions

• Evolution of tRNA modification systems:

  • E. sibiricum MiaA research contributes to understanding the conservation and divergence of tRNA modification pathways across diverse bacterial lineages

  • Structural studies of DMATase indicate homology to a class of small soluble kinases involved in nucleotide precursor biosynthesis, suggesting evolutionary relationships that may be further explored

  • Comparative genomics approaches can reveal how tRNA modification systems adapted to different environmental niches

• Structure-function relationships in tRNA-modifying enzymes:

  • The central channel architecture of DMATase, with separate entry points for tRNA and DMAPP substrates, represents a fascinating structural solution for bringing together two distinct substrates

  • Understanding how this architectural feature functions in E. sibiricum MiaA can provide insights into the catalytic mechanism and substrate specificity determinants

• Translation efficiency in extremophiles:

  • tRNA modifications significantly impact translation efficiency and accuracy

  • Research on E. sibiricum MiaA helps explain how psychrotolerant organisms maintain efficient translation at low temperatures

  • These insights may have broader implications for understanding cellular adaptation to extreme environments

Current research methodologies combining structural biology, enzyme kinetics, and comparative genomics provide complementary approaches to understanding how E. sibiricum MiaA contributes to the organism's ability to thrive in cold environments.

What potential biotechnological applications exist for recombinant E. sibiricum MiaA?

Recombinant E. sibiricum MiaA offers several promising biotechnological applications leveraging its unique properties as an enzyme from a psychrotolerant organism:

• Cold-active biocatalysis:

  • Development of cold-active enzymatic processes for industrial applications

  • Potential for energy savings in industrial biocatalysis by operating at lower temperatures

  • Application in temperature-sensitive reaction systems where elevated temperatures would be detrimental to substrate stability

• Synthetic biology applications:

  • Engineering of tRNA modifications in heterologous systems to modulate translation efficiency

  • Development of orthogonal tRNA modification systems for site-specific incorporation of non-canonical amino acids

  • Creation of synthetic tRNA populations with enhanced function at low temperatures

• Structural biology tools:

  • Use of the dimethylallyl modification as a site-specific probe for tRNA structure and function studies

  • Development of tRNA labeling strategies based on the enzymatic activity of MiaA

  • Application in cryo-EM studies where low-temperature stability of enzymes is advantageous

• Therapeutic potential:

  • Exploration of tRNA modifications as antibiotic targets

  • Development of specific inhibitors of bacterial tRNA modification enzymes

  • Investigation of tRNA modification in pathogenic bacteria adapted to the human body temperature

• Enzyme engineering platforms:

  • Using insights from E. sibiricum MiaA structure-function relationships to engineer enzymes with enhanced cold activity

  • Development of chimeric enzymes combining domains from psychrophilic and mesophilic homologs

  • Creation of biosensors based on conformational changes associated with substrate binding

The implementation of these applications requires detailed understanding of the enzyme's structure, function, and substrate specificities, areas where ongoing research on E. sibiricum MiaA continues to provide valuable insights.