Recombinant Kosmotoga olearia Methionyl-tRNA formyltransferase (fmt)

What is the functional role of Fmt in Kosmotoga olearia protein synthesis?

Fmt catalyzes the transfer of a formyl group from 10-formyldihydrofolate (10-CHO-DHF) or 10-formyltetrahydrofolate (10-CHO-THF) to the methionyl-tRNA$^{fMet}$ initiator molecule, ensuring proper initiation of bacterial translation . This reaction is essential for distinguishing initiator tRNA from elongator tRNA, a process critical for ribosomal fidelity. Methodologically, confirmatory assays involve LC-MS/MS quantification of dihydrofolate (DHF) by-products during in vitro formylation reactions, coupled with growth inhibition studies in ∆fmt mutants exposed to antifolates like trimethoprim (TMP) . For example, TMP sensitivity in wild-type K. olearia correlates with disrupted folate cycling, indirectly validating Fmt’s dependence on reduced folate pools .

What expression systems are optimal for producing recombinant K. olearia Fmt?

Heterologous expression in Escherichia coli BL21(DE3) with codon optimization is widely adopted due to its compatibility with thermophilic enzyme production . Key parameters include:

• Induction temperature: 18–25°C to minimize inclusion body formation.

• Vector selection: pET-based systems with N-terminal His-tags for immobilized metal affinity chromatography (IMAC) .
  Post-purification, validate enzymatic activity via radiolabeled formate incorporation assays, comparing kinetic parameters ($k_{cat}$, $K_m$) against native K. olearia extracts .

How does K. olearia Fmt substrate specificity differ from mesophilic homologs?

K. olearia Fmt uniquely utilizes 10-CHO-DHF, an oxidized folate derivative, in addition to 10-CHO-THF . This dual specificity was confirmed through competitive inhibition assays showing 10-CHO-DHF’s $K_i$ values comparable to 10-CHO-THF’s $K_m$. Structural predictions suggest a broader substrate-binding pocket accommodating bulkier folate derivatives, a hypothesis testable via crystallographic studies or molecular dynamics simulations.

How do transcriptional regulators in K. olearia modulate fmt expression under thermal stress?

Transcriptomic profiling of K. olearia at suboptimal (30°C) vs. optimal (65°C) temperatures revealed 573 differentially expressed genes, including fmt . At 77°C, fmt transcription decreases by 2.3-fold, correlating with upregulated heat shock proteins (e.g., DnaK, GroEL) . Investigate regulatory networks using chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors binding the fmt promoter under thermal stress. Comparative genomics highlights lateral gene transfer (LGT) events in K. olearia’s genome, including duplicated cold-shock proteins (Csps) that may stabilize fmt mRNA at low temperatures .

What structural motifs enable K. olearia Fmt’s thermostability?

Predicted α-helix-rich regions (residues 45–78, 112–135) and ionic interactions (e.g., Arg94–Glu121) contribute to thermostability . Validate these motifs via site-directed mutagenesis:

• R94A/E121A mutants: Assess melting temperature ($T_m$) shifts using differential scanning fluorimetry (DSF).

• Circular dichroism (CD) spectroscopy: Compare secondary structure integrity at 65°C vs. 30°C .
  Contradictory data may arise if mutations inadvertently disrupt folate-binding sites; thus, pair structural analyses with isothermal titration calorimetry (ITC) to monitor substrate affinity changes.

How do folate pool dynamics influence Fmt activity in vivo?

Antifolate treatments (e.g., TMP) deplete reduced folates (THF, 5,10-CH$_2$-THF) while elevating oxidized species (folic acid, DHF) . Quantify intracellular folate species via HPLC-coupled electrochemical detection and correlate with Fmt activity (Table 1):

Data adapted from demonstrate DHF’s inhibitory role, likely via competitive binding to Fmt’s active site.

Resolving discrepancies in Fmt’s optimal pH and temperature profiles

Early studies reported K. olearia Fmt activity peaks at pH 6.8 and 65°C , but later work observed residual activity at pH 5.5–8.0 and 20–80°C . To reconcile this:

• Standardize assay buffers: Use 50 mM HEPES (pH 6.8–7.2) vs. phosphate buffers (pH 5.5–6.5).

• Account for metabolite interference: DHF accumulation at low pH artificially suppresses activity .

• Assay duration: Short incubations (5–10 min) minimize enzyme denaturation at extremes.

Interpreting genomic variability in fmt homologs across Thermotogales

While K. olearia’s fmt shares 67% identity with Thermotoga maritima’s homolog, its flanking regions encode unique mobile elements (e.g., transposases, group II introns) . These elements may drive phase variation or differential expression across strains. To test this:

• Perform RNA-Seq on K. olearia cultures at 37°C vs. 65°C, quantifying fmt transcript levels .

• Use CRISPR interference (CRISPRi) to silence mobile elements and assess fmt expression stability .