Recombinant Agrobacterium vitis tRNA dimethylallyltransferase (miaA)

What is the biochemical function of tRNA dimethylallyltransferase (MiaA) in bacteria including Agrobacterium vitis?

MiaA functions as a tRNA prenyltransferase that catalyzes the addition of a prenyl group onto the N6-nitrogen of adenosine-37 within tRNAs that decode UNN codons. This creates i6A-37 tRNA, which is subsequently methylthiolated by MiaB to form ms2i6A-37. The bulky and hydrophobic ms2i6A-37 modification enhances tRNA interactions with target codons, promoting reading frame maintenance and translational fidelity. This enzyme represents a critical component of bacterial tRNA modification systems that ensure accurate protein synthesis.

The specificity of MiaA for tRNAs that recognize UNN codons means it plays a particularly important role in the translation of UNN-rich transcripts. Without proper MiaA-mediated modification, these transcripts are more susceptible to translational errors, including frameshifting and misreading .

How does MiaA differ from adenylate IPTs found in Agrobacterium species?

While both catalyze prenylation reactions, MiaA (a tRNA IPT) and adenylate IPTs (like Tmr and Tzs in A. tumefaciens) differ significantly in their substrates and cellular functions. MiaA specifically modifies tRNA-bound adenosine-37, affecting translation processes. In contrast, adenylate IPTs transfer prenyl groups to free nucleotides (primarily AMP in bacteria), producing cytokinins that function as plant growth regulators.

Biochemically, adenylate IPTs from Agrobacterium species prefer AMP as the prenyl chain acceptor and use both dimethylallyl pyrophosphate (DMAPP) and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) as prenyl chain donors. This contrasts with plant adenylate IPTs, which preferentially use ATP or ADP as acceptors . The HMBPP utilization allows direct production of trans-zeatin type cytokinins without requiring subsequent hydroxylation steps .

What is known about the evolutionary conservation of MiaA across bacterial species?

MiaA is remarkably conserved across bacterial species, reflecting its fundamental role in tRNA modification and translational regulation. The miaA genes are present in nearly all bacteria, with the notable exception of the genus Mycoplasma. The ms2i6A-37 modification catalyzed by the MiaA/MiaB pathway is conserved in both prokaryotes and eukaryotes, though the specific enzymes mediating this modification have diverged in evolutionarily distant organisms .

This conservation underscores the critical nature of proper tRNA modification for accurate translation across all life forms. In bacteria like E. coli, the absence of MiaA affects various cellular processes, including attenuation of amino acid operons, translation of stress response factors, DNA repair mechanisms, and spontaneous mutation frequencies . Similar functional importance would be expected in A. vitis, given the conserved nature of translational machinery.

What substrates are utilized by bacterial MiaA enzymes?

Bacterial MiaA enzymes utilize two key substrates: specific tRNAs and prenyl donors. The tRNA substrates must contain adenosine at position 37 and specifically decode UNN codons. For prenyl donors, MiaA can use both dimethylallyl pyrophosphate (DMAPP) and 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), with preferences varying between species and enzyme variants.

For experimental purposes, synthetic 17-base oligoribonucleotides that mimic the stem-loop region of tRNA can be used as substrates. Using such consensus oligoribonucleotides, studies of tRNA IPT from Nostoc sp. PCC 7120 (NoIPT2) demonstrated this enzyme utilizes both DMAPP and trans-HMBPP as donor substrates, with a strong preference for DMAPP . Similar studies would be valuable for characterizing the specific substrate preferences of A. vitis MiaA.

What expression systems are most effective for producing recombinant A. vitis MiaA?

For recombinant production of A. vitis MiaA, E. coli-based expression systems using vectors with inducible promoters provide an effective approach. Plasmids like pRR48 with tac promoters controlled by IPTG have been successfully employed for expressing MiaA and related enzymes in bacterial systems .

To optimize expression, consider these methodological approaches:

• Codon optimization based on the host organism's codon usage

• Addition of affinity tags to facilitate purification (C-terminal tags like Flag and 6xHis have been shown not to interfere with MiaA function)

• Testing different induction conditions (temperature, inducer concentration, duration)

• Exploring various E. coli strains, particularly those designed for expression of proteins with rare codons

For natural promoter studies, plasmids that incorporate the native A. vitis miaA promoter region can be constructed, similar to the approach used with pMiaA-Flagnat plasmids described for E. coli MiaA .

What purification strategies yield the highest enzymatic activity for recombinant MiaA?

Purification of recombinant MiaA with preserved enzymatic activity typically involves multiple chromatography steps with careful attention to buffer conditions. An effective purification strategy includes:

• Initial affinity chromatography using tags that don't compromise enzymatic activity (C-terminal Flag or His tags are preferred as they don't interfere with MiaA function)

• Secondary purification using size exclusion or ion exchange chromatography

• Utilization of buffers containing reducing agents (DTT or β-mercaptoethanol) to protect thiol groups

• Inclusion of glycerol (10-20%) to enhance protein stability during purification and storage

• Maintaining physiological pH (typically 7.0-8.0) throughout the purification process

Activity assays should be performed after each purification step to monitor enzyme functionality. Additionally, storage conditions (including flash freezing in liquid nitrogen versus slow freezing at -80°C) should be empirically tested to determine optimal approaches for maintaining long-term activity .

How can MiaA activity be quantitatively measured in vitro?

MiaA activity can be quantified through several complementary approaches:

• Radiometric assays: Using radiolabeled prenyl donors ([14C]-DMAPP or [3H]-DMAPP) to measure the incorporation of radioactive prenyl groups into tRNA substrates. This approach provides high sensitivity but requires specialized equipment for radiation detection.

• HPLC analysis: After the enzymatic reaction, tRNA substrates can be digested to nucleosides, and the resulting modified nucleosides (i6A or derivatives) can be separated and quantified by HPLC. This approach allows direct measurement of product formation.

• Mass spectrometry: LC-MS/MS analysis provides both identification and quantification of modified nucleosides with high specificity and sensitivity.

For kinetic characterization, reactions using varying concentrations of substrates can determine key parameters like Km and kcat. Related enzymes such as A. tumefaciens Tmr have shown Km values of 0.086 μM for AMP and 8.28 μM for DMAPP, with a kcat of 4.1 × 10−1 s−1 . Similar analyses would be valuable for characterizing A. vitis MiaA.

What controls should be included when studying MiaA-mediated tRNA modifications?

Rigorous experimental controls are essential when studying MiaA-mediated modifications:

• Enzyme controls:

  • Negative control using heat-inactivated enzyme

  • Catalytically inactive MiaA variants created through site-directed mutagenesis of key active site residues

  • Wild-type enzyme complementation in miaA knockout strains to verify functionality

• Substrate controls:

  • tRNAs lacking adenosine at position 37 (negative control)

  • Synthetic oligoribonucleotides mimicking the stem-loop region of tRNA

  • Comparison of different prenyl donors (DMAPP vs. HMBPP)

• Reaction condition controls:

  • Time-course analyses to ensure measurements are made within the linear range

  • Metal ion dependency tests (presence/absence of Mg2+ or Mn2+)

  • pH and temperature optimization experiments

• Downstream effect controls:

  • When studying translational effects, include controls for potential indirect effects on other cellular processes

  • Use dual-luciferase reporter systems with frameshift-prone sequences to directly measure translational fidelity

What amino acid residues are critical for the catalytic function of MiaA?

Based on structural and functional studies of related enzymes, several key amino acid residues are likely critical for A. vitis MiaA function:

• Hydrophilic residues in the reaction cavity: In A. tumefaciens Tzs (an adenylate IPT), His214 and Asp173 form a hydrophilic region crucial for substrate recognition and specificity. Mutations in these residues significantly affected both Km and kcat values, particularly for reactions with HMBPP .

• Positively charged residues for substrate binding: Lysine residues (such as Lys275 in related enzymes) play important roles in nucleotide substrate binding and orientation within the active site.

• Conserved motifs across MiaA homologs: Sequence alignments reveal conserved regions that likely contribute to the catalytic mechanism and substrate recognition.

The conservation of these residues varies among bacterial species, correlating with differences in substrate specificity. For example, A. tumefaciens enzymes maintain Lys at position 275, while Nostoc sp. NoIPT1 contains hydrophobic amino acids at the equivalent position, likely contributing to their different substrate preferences .

How do structural features of A. vitis MiaA influence substrate specificity between DMAPP and HMBPP?

The structural features that determine substrate specificity between DMAPP and HMBPP are primarily located within the active site of the enzyme. Based on studies of related enzymes, several key determinants can be identified:

• Hydrophilic versus hydrophobic active site residues: In A. tumefaciens Tzs, His214 and Asp173 create a hydrophilic region crucial for recognizing HMBPP, which contains a hydroxyl group. Enzymes retaining both hydrophilic charged residues show higher activity with HMBPP .

• Variability in substrate binding pocket: The presence of specific amino acids in the prenyl donor binding site correlates with substrate preferences. For instance, NoIPT1 from Nostoc contains substitutions (Y216 and V173) that alter its substrate preference compared to A. tumefaciens enzymes .

This structure-function relationship is illustrated by the observed activities with DMAPP and HMBPP, which correspond to the presence or absence of two hydrophilic charged residues in the active site. The table below shows kinetic parameters for related enzymes:

These differences in kinetic parameters reflect the structural variations in the substrate binding sites of these enzymes .

What mutations in MiaA affect its catalytic efficiency, and how might they be exploited experimentally?

Several types of mutations can affect MiaA catalytic efficiency, providing valuable experimental tools:

These mutations can be exploited experimentally in several ways:

• Creating catalytically inactive variants for control experiments

• Engineering MiaA variants with altered substrate preferences

• Developing MiaA mutants with enhanced activity or stability for biotechnological applications

• Constructing chimeric enzymes that combine domains from different MiaA homologs to study structure-function relationships

Site-directed mutagenesis guided by homology models based on related enzymes provides a powerful approach for systematic investigation of A. vitis MiaA structure-function relationships.

What is the kinetic mechanism of MiaA-catalyzed prenylation?

The kinetic mechanism of MiaA-catalyzed prenylation likely involves several discrete steps:

• Binding of tRNA substrate containing adenosine at position 37

• Binding of prenyl donor (DMAPP or HMBPP)

• Nucleophilic attack by the N6-nitrogen of adenosine-37 on the C1 of the prenyl donor

• Release of pyrophosphate

• Release of the modified tRNA containing i6A-37

While the exact order of substrate binding (random versus ordered sequential) has not been definitively established for A. vitis MiaA, studies of related enzymes suggest an ordered mechanism may be likely. Pre-steady-state kinetic analyses would be required to fully elucidate the microscopic steps involved in catalysis.

How does MiaA activity influence translational fidelity in bacteria?

MiaA-mediated tRNA modification significantly impacts translational fidelity through several mechanisms:

• Enhanced codon-anticodon interactions: The bulky and hydrophobic ms2i6A-37 modification strengthens the interaction between tRNAs and their cognate UNN codons, promoting accurate decoding .

• Reduced frameshifting: Properly modified tRNAs maintain reading frame more effectively. Both ablation and overproduction of MiaA stimulate translational frameshifting, indicating that balanced MiaA activity is critical for optimal translational fidelity .

• Differential effects on specific transcripts: UNN-rich genes are particularly susceptible to misreading in MiaA-deficient strains, creating a mechanism for selective translational regulation.

• Global proteome alterations: Changes in MiaA activity profoundly alter the bacterial proteome, affecting numerous cellular processes beyond direct translational effects .

These effects can be quantitatively assessed using dual-luciferase reporter systems containing frameshift-prone sequences. Such experimental approaches have demonstrated that MiaA functions like a rheostat, with both insufficient and excessive activity disrupting optimal translation .

What is the relationship between MiaA and bacterial virulence?

MiaA plays a crucial role in bacterial virulence through multiple mechanisms:

• Stress adaptation: MiaA is essential for bacterial adaptation to stressors encountered during infection, including oxidative stress, nutrient limitation, and osmotic pressure. In extraintestinal pathogenic E. coli (ExPEC), MiaA deletion mutants showed impaired growth under various stress conditions .

• Motility regulation: MiaA-deficient strains demonstrated reduced motility in swim assays, a phenotype often correlated with decreased virulence in many bacterial pathogens .

• Host cell interactions: In cell culture models, MiaA contributes to bacterial invasion and intracellular persistence within host cells .

• Competitive fitness: MiaA enhances bacterial competitive fitness in mixed cultures, an important factor during establishment of infection in host environments .

• Virulence factor expression: By affecting the bacterial proteome, MiaA influences the expression of numerous virulence factors and regulatory proteins that coordinate pathogenesis.

These findings highlight MiaA as a potential target for antimicrobial development, as inhibiting this enzyme could potentially attenuate bacterial virulence without directly killing the pathogen, potentially reducing selective pressure for resistance development.

How does environmental stress modulate MiaA expression and activity?

Environmental stresses trigger sophisticated modulation of MiaA expression and activity:

• Post-transcriptional regulation: In ExPEC, MiaA levels shifted in response to stress via post-transcriptional mechanisms, resulting in marked changes in the amounts of fully modified MiaA substrates .

• Stress-specific responses: Different stressors (oxidative damage, nutrient limitation, osmotic pressure) may elicit distinct patterns of MiaA regulation, allowing fine-tuned responses to specific environmental challenges.

• Integration with global stress responses: MiaA regulation appears integrated with broader stress response networks, including stationary phase adaptation systems. In E. coli, MiaA impacts translation of the stationary phase sigma factor RpoS and the small RNA chaperone Hfq .

• Metabolic precursor availability: Stress conditions may alter the availability of prenyl donors (DMAPP or HMBPP), creating an additional layer of regulation through substrate limitation.

This dynamic regulation allows bacteria to rapidly adjust their translational machinery to adapt to challenging environments, representing a novel aspect of stress response that may be conserved across bacterial species including A. vitis. MiaA acts much like a rheostat that can be used to realign global protein expression patterns to optimize cellular responses to environmental conditions .

What phenotypic changes result from MiaA deletion or overexpression?

Both deletion and overexpression of MiaA produce significant and distinct phenotypic changes:

MiaA deletion typically results in:

• Increased sensitivity to environmental stressors (high salt, oxidative damage, specific amino acids)

• Reduced motility in swim assays

• Impaired competitive fitness in mixed cultures

• Decreased virulence in infection models

• Altered proteome composition with specific effects on UNN-rich transcripts

• Elevated translational frameshifting

• Inability to effectively resolve aberrant DNA-protein crosslinks

• Somewhat elevated spontaneous mutation frequencies

MiaA overexpression leads to:

• Paradoxically increased translational frameshifting, similar to deletion mutants

• Altered proteome composition distinct from both wild-type and deletion mutants

• Potential metabolic imbalances due to overconsumption of prenyl donor substrates

• Changes in stress response mechanisms

The bidirectional impact of MiaA levels on translational fidelity underscores the importance of precisely regulated MiaA activity. This suggests MiaA functions as a regulatory nexus that must be maintained within an optimal range for proper cellular function .

What structural and functional differences exist between bacterial MiaA and tRNA IPTs in other organisms?

Bacterial MiaA and tRNA IPTs in other organisms exhibit several key structural and functional differences:

How does substrate utilization of A. vitis MiaA compare with other bacterial tRNA IPTs?

Substrate utilization can vary significantly among bacterial tRNA IPTs, particularly regarding prenyl donors:

• Prenyl donor preferences: Some bacterial MiaAs preferentially use DMAPP, while others efficiently utilize HMBPP. These preferences correlate with specific amino acid residues in the active site. The presence of hydrophilic residues (like His214 and Asp173 in A. tumefaciens Tzs) often correlates with efficient HMBPP utilization .

• tRNA substrate specificity: While all bacterial MiaAs modify tRNAs with adenosine at position 37, subtle differences may exist in recognition efficiency for different tRNA species.

• Kinetic parameters: The Km and kcat values can vary substantially between species. For instance, comparing A. tumefaciens Tmr (kcat = 4.1 × 10−1 s−1 with AMP) and FasD (kcat = 7.9 × 10−3 s−1 with AMP) reveals significant differences in catalytic efficiency .

In Nostoc sp. PCC 7120, tRNA IPT (NoIPT2) uses both DMAPP and trans-HMBPP as donor substrates, with a strong preference for DMAPP . Without specific biochemical characterization, the precise substrate preferences of A. vitis MiaA remain to be determined, though insights could be gained through sequence analysis and homology modeling based on characterized enzymes.

What methodological approaches best reveal species-specific differences in MiaA function?

Several complementary methodological approaches can effectively reveal species-specific differences in MiaA function:

• Comparative biochemistry:

  • Side-by-side kinetic analysis of recombinant MiaA enzymes from different species

  • Substrate preference studies comparing utilization of DMAPP versus HMBPP

  • Temperature and pH optima determination to identify adaptation to specific environmental niches

• Structural biology:

  • X-ray crystallography or cryo-EM studies of MiaA enzymes from different species

  • Computational modeling and molecular dynamics simulations to identify species-specific structural features

  • Structure-guided mutagenesis to test the functional importance of divergent residues

• Cross-species complementation:

  • Expressing MiaA from different species in a common MiaA-deficient host

  • Quantitative assessment of complementation efficiency using growth, stress resistance, and translational fidelity assays

  • Construction of chimeric enzymes combining domains from different species to identify functional determinants

• Systems biology:

  • Comparative transcriptomics and proteomics of wild-type and MiaA mutants across species

  • Global analysis of tRNA modification profiles to identify species-specific patterns

  • Network analysis to understand species-specific integration of MiaA function with other cellular processes

These approaches would reveal both conserved core functions and species-specific adaptations of MiaA enzymes, providing insights into the evolution of this critical tRNA modification system and potentially identifying species-specific vulnerabilities that could be targeted for antimicrobial development .