Recombinant Finegoldia magna Methionyl-tRNA formyltransferase (fmt)

How does the folate pathway interact with Fmt activity?

The folate pathway plays a critical role in supporting Fmt activity through the production of formyl donor substrates. This interconnection involves several key enzymes and metabolites:

• Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, converts 5,10-methylene tetrahydrofolate (5,10-CH2-THF) to 5,10-methenyltetrahydrofolate (5,10-CH+-THF) through its dehydrogenase activity, and subsequently converts 5,10-CH+-THF to 10-formyltetrahydrofolate (10-CHO-THF) through its cyclohydrolase activity .

• 10-CHO-THF serves as the primary formyl donor for Fmt to formylate Met-tRNAfMet .

• Dihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF), as well as the reduction of 10-formyldihydrofolate (10-CHO-DHF) to 10-formyltetrahydrofolate (10-CHO-THF) .

• Antifolate drugs like trimethoprim (TMP) target DHFR, affecting the availability of reduced folate species and consequently impacting Fmt function .

What methods are recommended for detecting and analyzing Fmt activity in vitro?

Several complementary methodologies can be employed to detect and analyze Fmt activity in vitro:

In vitro formylation assay

The recommended protocol involves:

• Preparing deacylated tRNAfMet (can be isolated from Δfmt strains overexpressing tRNAfMet)

• Charging tRNAfMet with methionine using purified Methionyl-tRNA synthetase (MetRS)

• Incubating Met-tRNAfMet with different folate species (e.g., 10-CHO-THF, 10-CHO-DHF) and purified Fmt

• Analyzing the reaction products using one of the methods described below

Northern blotting for tRNA analysis

Northern blotting can effectively distinguish between formylated and unformylated tRNAfMet:

• Prepare total tRNAs under cold and acidic conditions to preserve the ester bond linking amino acids to tRNA

• Treat samples with CuSO4 (for deacylation of Met-tRNAfMet) or with high pH buffer (for deacylation of both fMet-tRNAfMet and Met-tRNAfMet)

• Separate tRNAs on acid urea PAGE

• Perform Northern blotting using 5'-32P end-labeled DNA oligomers complementary to tRNAfMet

• Analyze by phosphor-imaging

LC-MS/MS analysis of reaction products

For detailed identification of reaction products:

• Perform the formylation reaction as described above

• Stop the reaction with acidification and heat treatment

• Process the sample with folate gamma-glutamyl hydrolase to produce mono-glutamylated folate species

• Filter through a low molecular weight cut-off filter

• Analyze by LC-MS/MS using a C18 reversed-phase column

• Monitor specific m/z values for various folate species:

  • Folic acid (m/z 440.1324)

  • DHF (m/z 442.1783)

  • THF (m/z 444.1600)

  • 10-CHO-folic acid (m/z 468.1272)

  • 10-CHO-DHF (m/z 470.1400)

  • 10-CHO-THF/5-CHO-THF (m/z 472.1580)

  • 5,10-CH+-THF (m/z as appropriate)

What expression systems are most effective for producing recombinant F. magna Fmt?

Based on established protocols for Fmt expression, the following approaches are recommended:

Expression vectors and host strains

• Vector selection: pET28b or similar expression vectors with a 6×His-tag for purification are optimal

• Bacterial hosts: E. coli BL21(DE3) or Rosetta(DE3) pLysS strains are preferred, with the latter being especially useful if F. magna uses rare codons

Expression conditions optimization

• Induction: 0.1-0.5 mM IPTG at mid-log phase (OD600 ~0.6)

• Temperature: Lower temperatures (16-20°C) often yield higher amounts of soluble protein

• Duration: 4-16 hours post-induction, depending on temperature

• Media supplements: Consider adding extra zinc or other cofactors if required for proper folding

Purification strategy

• Affinity chromatography: Nickel-NTA for His-tagged protein

• Ion exchange chromatography: For further purification if needed

• Size exclusion chromatography: To remove aggregates and ensure homogeneity

• Buffer optimization: Include reducing agents (DTT or β-mercaptoethanol) to maintain enzyme activity

How does F. magna Fmt utilize 10-formyldihydrofolate as an alternative substrate?

Recent research has revealed that Fmt can utilize 10-CHO-DHF as an alternative substrate to the canonical 10-CHO-THF. This discovery has significant implications for understanding bacterial metabolism and antibiotic resistance.

Experimental evidence for 10-CHO-DHF utilization

In vitro studies have demonstrated that:

• The formyl group from 10-CHO-DHF can be effectively transferred to Met-tRNAfMet by purified Fmt

• This reaction produces DHF as a by-product, which has been verified by LC-MS/MS analysis

• The formylation reaction with 10-CHO-DHF as substrate is efficient enough to support bacterial protein synthesis

Physiological significance

This alternative substrate utilization has important physiological implications:

What experimental approaches can be used to study substrate specificity of F. magna Fmt?

To comprehensively investigate the substrate specificity of F. magna Fmt, researchers should consider multiple complementary approaches:

Kinetic analysis

• Steady-state kinetics:

  • Determine Km, kcat, and kcat/Km values for different substrates (10-CHO-THF, 10-CHO-DHF)

  • Compare kinetic parameters to assess substrate preference

  • Evaluate the effects of pH, temperature, and ionic strength on substrate utilization

• Pre-steady-state kinetics:

  • Use rapid kinetic techniques (stopped-flow, quench-flow) to measure transient kinetic parameters

  • Identify rate-limiting steps in the catalytic cycle with different substrates

Structural biology approaches

• X-ray crystallography or cryo-EM:

  • Determine the structure of F. magna Fmt alone and in complex with different substrates

  • Identify key residues involved in substrate recognition and catalysis

  • Compare with known structures of Fmt from other organisms

• Site-directed mutagenesis:

  • Target specific residues predicted to be involved in substrate binding

  • Evaluate the effects of mutations on activity with different substrates

  • Create structure-function correlations

Competition assays

Design assays where both 10-CHO-THF and 10-CHO-DHF are present, and:

• Vary the ratio of substrates to determine preference

• Measure formylation rates and product distribution

• Analyze the results to calculate relative substrate specificities

How do antifolate drugs affect Fmt function and bacterial translation?

Antifolate drugs, particularly those targeting DHFR like trimethoprim, have complex effects on Fmt function and bacterial translation:

Differential sensitivity based on Fmt status

Experimental evidence shows that:

• FolD-deficient mutants exhibit increased sensitivity to trimethoprim

• Fmt over-expressing strains show greater sensitivity to trimethoprim than Δfmt strains

• This suggests that high Fmt activity in the presence of antifolates can be detrimental to bacterial growth

What methods are optimal for studying Fmt-tRNA interactions?

Investigating the specific interactions between F. magna Fmt and tRNAfMet requires specialized techniques:

RNA structural and interaction analysis

• RNA footprinting:

  • Use chemical or enzymatic probes to identify nucleotides protected by Fmt binding

  • Compare footprinting patterns with free tRNAfMet and tRNAfMet-Fmt complexes

• Electrophoretic mobility shift assays (EMSA):

  • Titrate increasing amounts of Fmt with labeled tRNAfMet

  • Quantify binding affinities under various conditions

  • Compare wild-type tRNAfMet with mutant variants

• Isothermal titration calorimetry (ITC):

  • Measure thermodynamic parameters of binding (ΔH, ΔS, ΔG)

  • Determine stoichiometry and binding constants

Structural approaches

• Cryo-EM analysis of Fmt-tRNAfMet complexes:

  • Visualize the three-dimensional arrangement of the complex

  • Identify key interaction points between protein and RNA

• Chemical crosslinking followed by mass spectrometry:

  • Identify specific contact points between Fmt and tRNAfMet

  • Map the binding interface at amino acid and nucleotide resolution

tRNA mutational analysis

• Generate tRNAfMet variants with mutations in potential recognition elements

• Assess the impact on:

  • Binding affinity using methods described above

  • Formylation efficiency in vitro

  • Translation initiation fidelity in vivo (if possible)

How can metabolomic approaches enhance Fmt research?

Metabolomic techniques offer powerful tools for understanding the broader impact of Fmt function on cellular metabolism:

LC-MS/MS profiling of folate pathway metabolites

• Comprehensive analysis of folate species:

  • Extract and analyze all relevant folate metabolites (THF, DHF, 10-CHO-THF, 10-CHO-DHF, etc.)

  • Monitor shifts in folate pool composition under different conditions

  • Track the formation of reaction products and intermediates

• Experimental design considerations:

  • Compare wild-type vs. fmt mutant strains

  • Analyze effects of antifolate treatments at different concentrations

  • Examine metabolite profiles across growth phases

Application to antifolate resistance studies

Metabolomic approaches can reveal how bacteria respond metabolically to antifolate stress:

• Monitor changes in folate pathway metabolites during antifolate exposure

• Compare responses in strains with different Fmt expression levels

• Identify potential metabolic biomarkers of resistance development

What are the main challenges in expressing and purifying active recombinant F. magna Fmt?

Researchers should be aware of several challenges when working with recombinant F. magna Fmt:

Expression challenges

• Codon usage: F. magna and E. coli have different codon preferences, potentially requiring codon optimization or use of strains supplemented with rare tRNAs

• Protein folding: Maintaining proper folding may require expression at lower temperatures (16-20°C)

• Solubility: The protein may form inclusion bodies, necessitating:

  • Optimization of induction conditions (lower IPTG, temperature)

  • Use of solubility-enhancing fusion tags (SUMO, MBP, etc.)

  • Co-expression with chaperones if needed

Purification considerations

• Maintaining activity: Fmt may be sensitive to oxidation, requiring reducing agents in all buffers

• Protein stability: Consider adding glycerol (10-20%) and appropriate salt concentrations

• Aggregation prevention: Include low concentrations of detergents if necessary

• Storage conditions: Test stability at different temperatures (-80°C, -20°C, 4°C) and with various cryoprotectants

How can the purity and activity of recombinant F. magna Fmt be verified?

A multi-faceted approach to quality control is essential:

Purity assessment

• SDS-PAGE analysis:

  • Visualize protein purity with Coomassie or silver staining

  • Estimate purity percentage using densitometry

• Size exclusion chromatography:

  • Evaluate homogeneity and detect aggregates or degradation products

  • Determine oligomeric state (monomer, dimer, etc.)

• Mass spectrometry:

  • Confirm protein identity and molecular weight

  • Detect post-translational modifications or truncations

Activity verification

• Enzymatic assays:

  • Measure formylation activity using methods described in section 1.3

  • Compare specific activity with published values for other Fmt enzymes

• Functional tests:

  • Complementation assays in Fmt-deficient strains

  • In vitro translation using purified translation components

How might F. magna Fmt be exploited as a potential antimicrobial target?

Given the critical role of Fmt in bacterial translation initiation, it represents a promising antimicrobial target:

Rational inhibitor design strategies

• Structure-based drug design:

  • Target the active site or substrate binding pockets

  • Design competitive inhibitors that mimic substrate structure

  • Exploit species-specific structural features for selectivity

• Allosteric inhibition:

  • Identify regulatory or allosteric sites

  • Design molecules that lock the enzyme in inactive conformations

Combination therapy approaches

• Fmt inhibitors with antifolates:

  • Target both Fmt and DHFR simultaneously

  • Exploit the synergistic vulnerability created by the interconnection between these enzymes

  • Potentially overcome existing resistance mechanisms

• Translation-targeting combinations:

  • Combine Fmt inhibitors with other antibiotics targeting different aspects of translation

  • Achieve synergistic effects and reduce resistance development

What is the relationship between bacterial microbiome composition and Fmt activity in human health?

While the search results don't directly address this question, several connections can be made:

Potential impact on urogenital microbiome

• F. magna is known to be part of the human urogenital microbiome, as are other bacteria with active Fmt systems

• The microbiome composition may be influenced by differences in Fmt activity and efficiency

• Antifolate drugs may selectively impact bacteria based on their Fmt activity and folate metabolism

Research opportunities

• Metagenomic analysis:

  • Compare fmt gene sequences and expression levels across microbiome populations

  • Correlate with microbiome stability and response to antifolates

• Metabolomic studies:

  • Analyze folate pathway metabolites in microbiome samples

  • Investigate correlations between folate profiles and microbiome composition