Recombinant Leptothrix cholodnii Methionyl-tRNA formyltransferase (fmt)

Overview of Recombinant Leptothrix cholodnii Methionyl-tRNA Formyltransferase (Fmt)

Recombinant Leptothrix cholodnii Methionyl-tRNA formyltransferase (Fmt) is a genetically engineered enzyme critical for bacterial translation initiation. This enzyme catalyzes the formylation of methionyl-tRNA (Met-tRNA) to formylmethionyl-tRNA (fMet-tRNA), a key step enabling proper ribosome binding and initiation of protein synthesis in prokaryotes . The recombinant form is produced for research applications, particularly in studies of bacterial translation fidelity and antibiotic targeting .

Domain Architecture

Fmt comprises two domains:

• N-terminal Rossmann-fold domain: Binds the formyl donor 10-formyltetrahydrofolate (10-CHO-THF) or its analog 10-formyldihydrofolate (10-CHO-DHF) .

• C-terminal OB-fold β-barrel domain: Facilitates tRNA binding through positively charged residues .

Catalytic Mechanism

Fmt transfers a formyl group from 10-CHO-THF/DHF to the methionine moiety of Met-tRNA, producing fMet-tRNA and dihydrofolate/tetrahydrofolate as byproducts .

Substrate Flexibility

• Fmt utilizes both 10-CHO-THF and 10-CHO-DHF as formyl donors in vitro, expanding its functional repertoire under folate-limiting conditions .

• LC-MS/MS confirmed dihydrofolate (DHF) as a reaction byproduct when using 10-CHO-DHF .

Antibiotic Sensitivity Link

• Overexpression of Fmt increases bacterial susceptibility to trimethoprim (TMP), a folate biosynthesis inhibitor .

• Δfmt mutants show resistance to TMP, highlighting Fmt’s role in folate pool homeostasis .

Applications in Biotechnology

1. Translation Initiation Studies: Used to investigate tRNA formylation’s role in start-codon selection .

2. Antibiotic Development: Target for compounds disrupting folate metabolism in pathogenic bacteria .

3. Protein Engineering: Platform for modifying formylation efficiency via domain-swapping experiments .

Technical Considerations for Experimental Use

• Reaction Conditions: Optimal activity achieved with 25 µM 10-CHO-THF/DHF and 0.2 µg enzyme in aminoacylation buffer (pH 7.5) .

• Activity Assay: Formylation reaction terminated using 0.1 M HCl and β-mercaptoethanol, followed by centrifugation at 15,800×g .

Research Gaps and Future Directions

• No in vivo functional studies of L. cholodnii Fmt are reported despite its recombinant availability .

• Potential interactions with other folate-dependent enzymes (e.g., PurU, ArnA) remain unexplored in this species .

• Structural data for the native L. cholodnii enzyme is lacking compared to E. coli homologs .