Recombinant Frankia sp. tRNA dimethylallyltransferase (miaA)

Enzymatic Function and Biological Role

MiaA is a conserved tRNA-modifying enzyme belonging to the prenyltransferase family (EC 2.5.1.75). Its catalytic activity involves two steps:

1. Substrate binding: MiaA first binds tRNA, positioning A37 for modification .

2. Prenylation: A dimethylallyl group is transferred from DMAPP to the N⁶-position of A37, forming i⁶A37 .

In Frankia sp., this modification is essential for optimizing translational efficiency under stress conditions, akin to its role in pathogenic E. coli (ExPEC), where MiaA deficiency impairs fitness, virulence, and stress adaptation .

Biochemical Properties

Recombinant Frankia sp. MiaA is typically expressed in E. coli or yeast systems, yielding >85% purity via SDS-PAGE . Key properties include:

Functional Insights from Homologs

Studies on E. coli MiaA provide critical insights into its regulatory roles:

• Proteome modulation: MiaA levels influence frameshifting rates and global protein expression, acting as a "rheostat" for stress adaptation .

• Virulence: In ExPEC, MiaA is crucial for host-cell invasion and intracellular persistence .

• Post-transcriptional regulation: MiaA deficiency reduces RpoS (σ^S) levels, impairing stationary-phase adaptation .

Applications and Research Tools

Recombinant Frankia sp. MiaA is utilized in:

• Mechanistic studies: Elucidating tRNA modification pathways and translational fidelity .

• Biotechnology: Engineering stress-resistant microbial strains via tRNA modification.

• Drug discovery: Targeting MiaA in pathogenic bacteria (e.g., urinary tract infection pathogens) .

Expression and Purification Protocols

Standard protocols for recombinant MiaA production include:

1. Cloning: miaA gene insertion into plasmids (e.g., pBAD24 or pRR48) under inducible promoters .

2. Expression: Induction with IPTG or arabinose in E. coli BL21(DE3) .

3. Purification: Affinity chromatography (e.g., His-tag systems) followed by gel filtration .

Future Directions

Unresolved questions include:

• Structural dynamics: How tRNA binding induces conformational changes in MiaA .

• Host-specific roles: Functional divergence of MiaA in Frankia sp. versus pathogens .

• Therapeutic potential: Developing MiaA inhibitors to combat antibiotic-resistant infections .