Recombinant Escherichia coli Methionyl-tRNA formyltransferase (fmt)
Description
Enzymatic Mechanism and Substrate Specificity
FMT transfers a formyl group from N<sup>10</sup>-formyltetrahydrofolate to the α-amino group of methionyl-tRNA<sup>fMet</sup>. Key specificity determinants include:
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Acceptor stem of tRNA<sup>fMet</sup>: Mutations in this region (e.g., G72G73) reduce formylation efficiency by >90% .
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Catalytic residues: Conserved Asn-106, His-108, and Asp-144 facilitate formyl group transfer .
Table 2: Kinetic Parameters of Wild-Type vs. Mutant FMT
| Substrate tRNA | k<sub>cat</sub> (min<sup>-1</sup>) | K<sub>m</sub> (μM) | Catalytic Efficiency (k<sub>cat</sub>/K<sub>m</sub>) |
|---|---|---|---|
| Wild-type tRNA<sup>fMet</sup> | 12.4 ± 1.2 | 0.52 ± 0.06 | 23.8 |
| U35A36/G72G73 mutant | 0.8 ± 0.1 | 15.3 ± 2.1 | 0.05 |
| G41R FMT mutant | 21.7 ± 2.5 | 0.82 ± 0.09 | 26.5 (for U35A36/G72G73 tRNA) |
Data from suppressor mutation studies showing Gly41→Arg/Lys mutants restore activity for defective tRNAs .
Functional Role in Protein Synthesis
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Initiation complex formation: Formylation ensures recognition by initiation factor IF2 and prevents elongation factor binding .
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Cellular growth dependency: E. coli strains with disrupted fmt exhibit severe growth defects (60% reduced growth rate at 37°C) .
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Cross-species functionality: Recombinant FMT expressed in Saccharomyces cerevisiae formulates 70% of cytoplasmic initiator tRNA, causing slow growth reversible by co-expressing deformylase .
Key Mutations and Phenotypic Effects:
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Gly41→Arg/Lys: Restores formylation of acceptor-stem-defective tRNAs, increasing catalytic efficiency 26-fold .
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C-terminal deletions (Δ18–80 aa): Reduce activity by >95%, underscoring the domain’s role in tRNA binding .
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Linker region lysine substitutions: Decrease k<sub>cat</sub> by 5–10 fold, highlighting charge-dependent tRNA interactions .
Recombinant Expression and Applications
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Expression systems: Successfully produced in E. coli and S. cerevisiae, with yeast-expressed FMT retaining 70% activity .
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Biotechnological utility: Used in studies of translation initiation mechanisms and antibiotic targeting (e.g., deformylase inhibitors) .
Evolutionary and Functional Variability
While essential in E. coli, FMT is dispensable in some eubacteria, suggesting evolutionary divergence in translation initiation strategies . The enzyme’s conservation across prokaryotes makes it a target for broad-spectrum antimicrobial agents.
Product Specs
Target Background
KEGG: eck:EC55989_3704
