Recombinant Desulfovibrio vulgaris Methionyl-tRNA formyltransferase (fmt)

What is the primary function of Methionyl-tRNA Formyltransferase in bacterial systems?

Methionyl-tRNA Formyltransferase (fmt) catalyzes the formylation of methionyl-tRNA, which irreversibly commits methionyl-tRNAfMet to the initiation of translation in eubacteria . This critical enzymatic reaction transfers a formyl group from 10-formyltetrahydrofolate (FTHF) to the amino group of the methionine moiety esterified to the initiator tRNA. The formylation process is essential for proper protein synthesis initiation, as underscored by observations that inactivation of the formylase gene in Escherichia coli severely impairs cell growth . The formylated initiator tRNA is specifically recognized by initiation factor IF2, preventing its interaction with elongation factor EF-Tu and thereby ensuring proper channeling toward the ribosomal P-site.

How does the domain structure of bacterial Methionyl-tRNA Formyltransferase contribute to its catalytic function?

Bacterial Methionyl-tRNA Formyltransferases typically contain two distinct domains connected by an elongated linker region . The N-terminal domain (approximately residues 1-189 in E. coli) harbors the catalytic center and contains a Rossmann fold that shares structural similarity with glycinamide ribonucleotide transformylase (GARF), which also utilizes 10-formyltetrahydrofolate as a formyl donor . The C-terminal domain (residues 209-314 in E. coli) consists of a β-barrel structure that plays a crucial role in tRNA recognition. During catalysis, both domains interact with the L-shaped tRNA molecule, with the acceptor arm of tRNA clamped between one side of the C-terminal domain and a large loop (loop 1, residues 34-49) in the catalytic domain . This domain architecture enables precise positioning of the 3' end of the initiator tRNA within the active site for the formylation reaction.

What structural features of initiator tRNA are specifically recognized by Methionyl-tRNA Formyltransferase?

The primary structural determinant for specific recognition of initiator tRNA by Methionyl-tRNA Formyltransferase is the absence of base pairing at the top of the acceptor helix, characterized in E. coli initiator tRNA as a C1-A72 mismatch . Additional recognition elements include the A73 discriminator base and the specific base pairs G2-C71, C3-G70, and G4-C69 in the acceptor arm . Crystal structure analysis of E. coli formylase complexed with formyl-methionyl-tRNAfMet demonstrates that the enzyme wedges a loop into the major groove of the acceptor helix, causing the C1-A72 mismatch to split and the 3' arm to bend inside the active center . This recognition mechanism is fundamentally different from that employed by elongation factor Tu, which binds the acceptor arm of aminoacylated elongator tRNAs on the T-stem side.

What are the optimal expression systems for producing recombinant D. vulgaris Methionyl-tRNA Formyltransferase?

For optimal expression of recombinant D. vulgaris Methionyl-tRNA Formyltransferase, researchers should consider a methodological approach similar to that used for other bacterial formylases. Based on established protocols, E. coli BL21(DE3) or equivalent strains carrying pET-derived expression vectors with appropriate antibiotic resistance markers represent effective expression systems. The recombinant construct should incorporate a purification tag (such as His6 or GST) to facilitate subsequent purification steps.

The expression methodology typically involves:

• Transformation of the expression construct into the chosen E. coli strain

• Culture growth at 37°C in rich medium (such as LB) to an OD600 of 0.6-0.8

• Temperature reduction to 18-25°C prior to induction

• Induction with 0.1-0.5 mM IPTG

• Post-induction expression for 4-16 hours at reduced temperature

This approach minimizes inclusion body formation while maximizing the yield of soluble, correctly folded enzyme. For D. vulgaris fmt specifically, researchers may need to optimize codon usage for expression in E. coli or consider alternative expression hosts if toxicity issues arise.

What purification strategy yields the highest activity of recombinant D. vulgaris fmt?

A multi-step purification strategy typically yields the highest activity for recombinant formyltransferases. Based on biochemical properties shared among bacterial formylases, the following methodological approach is recommended:

• Initial capture using affinity chromatography (Ni-NTA for His-tagged constructs)

• Buffer exchange to remove imidazole using dialysis or gel filtration

• Ion exchange chromatography (typically Q-Sepharose or SP-Sepharose depending on the isoelectric point)

• Size exclusion chromatography as a polishing step

Throughout purification, buffers should contain:

• 20-50 mM Tris or HEPES pH 7.5-8.0

• 100-200 mM KCl or NaCl

• 1-10 mM 2-mercaptoethanol or DTT

• 5-10% glycerol for stability

• Potentially 0.1 mM EDTA to chelate metal ions that might interfere with activity

The purification progress should be monitored using both SDS-PAGE for purity assessment and activity assays to identify fractions with the highest specific activity. Typical yields reported for recombinant bacterial formylases range from 5-20 mg of pure protein per liter of culture.

How does the crystal structure of D. vulgaris fmt compare to E. coli fmt?

While crystal structures of E. coli fmt have been well characterized, including the complex with formyl-methionyl-tRNAfMet at 2.8 Å resolution , specific structural data for D. vulgaris fmt remains limited. Based on sequence homology and conserved catalytic mechanisms among bacterial formyltransferases, researchers can make informed predictions about structural similarities and differences.

For comparative structural analysis between D. vulgaris and E. coli fmt, researchers should employ the following methodological approaches:

• Generate high-quality crystals of purified recombinant D. vulgaris fmt using vapor diffusion techniques, screening various precipitants, pH values, and additives

• Collect X-ray diffraction data at synchrotron sources with resolutions preferably better than 2.5 Å

• Solve the structure using molecular replacement with E. coli fmt (PDB ID: 2FMT) as a search model

• Perform rigorous refinement and validation of the structural model

• Analyze structural differences particularly in:

  • The catalytic N-terminal domain containing the Rossmann fold

  • The idiosyncratic loop 1 (residues 34-49 in E. coli) involved in tRNA recognition

  • The β-barrel C-terminal domain

  • The linker region connecting the two domains

Such comparative structural analysis would provide insights into potential adaptations of D. vulgaris fmt to the organism's unique physiological environment.

What residues constitute the active site of D. vulgaris fmt and how do they compare to those in other bacterial formyltransferases?

The active site residues of bacterial formyltransferases are generally highly conserved, reflecting their essential catalytic function. In E. coli fmt, key active site residues include Asn108, which interacts with the formyl group, and hydrophobic residues including Phe14, Ile123, Leu136, Leu171, Ala89, Pro122, and Tyr168, which form a cavity accommodating the methionine side chain .

For comprehensive identification of active site residues in D. vulgaris fmt, researchers should employ:

• Sequence alignment with well-characterized bacterial formyltransferases to identify potentially conserved catalytic residues

• Homology modeling based on the E. coli fmt crystal structure (if structural data for D. vulgaris fmt is unavailable)

• Site-directed mutagenesis of predicted active site residues followed by kinetic analysis to confirm their role in catalysis

• Structural analysis via X-ray crystallography or cryo-EM, ideally with bound substrate analogs or inhibitors

The specific hydrophobic character of the methionine-binding pocket in fmt enzymes accounts for their ability to formylate initiator tRNAs misesterified with other amino acids, albeit with reduced efficiency .

What is the optimal biochemical assay for measuring D. vulgaris fmt activity?

The optimal assay for measuring D. vulgaris fmt activity would be similar to established methodologies for bacterial formyltransferases. A comprehensive enzyme activity assay should include:

• Reaction components:

  • Purified recombinant D. vulgaris fmt (0.1-10 nM)

  • Met-tRNAfMet as substrate (0.05-15 μM range for kinetic studies)

  • 10-formyltetrahydrofolate (FTHF) as formyl donor (125-200 μM)

  • Buffer composition: 20 mM Tris-HCl pH 7.6, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 150 mM KCl, 7 mM MgCl₂

• Detection methods:

  • Radiometric assay using [35S]-Met-tRNAfMet and quantifying formylated product through acid precipitation and scintillation counting

  • HPLC separation of formylated and non-formylated Met-tRNAfMet

  • Mass spectrometry analysis of reaction products

• Kinetic analysis:

  • Determination of Km and kcat values for both tRNA and FTHF substrates

  • Analysis of temperature and pH optima

  • Assessment of divalent cation requirements

This methodological approach allows for precise quantification of enzymatic activity and comparative analysis with other bacterial formyltransferases.

How does the substrate specificity of D. vulgaris fmt compare with formyltransferases from other bacterial species?

Substrate specificity analysis for D. vulgaris fmt requires systematic comparison with other bacterial formyltransferases using identical experimental conditions. Based on studies with E. coli fmt, the following methodological approach is recommended:

• Substrate preparation:

  • Express and purify initiator tRNAs from different bacterial species with various acceptor stem sequences

  • Generate tRNA variants with specific mutations, particularly focusing on positions 1, 72, 73, and base pairs in the acceptor stem

  • Aminoacylate these tRNAs with methionine using purified methionyl-tRNA synthetase

• Comparative activity analysis:

  • Determine relative kcat/Km values for each tRNA substrate

  • Express results as percentages relative to the cognate wild-type initiator tRNA

• Specificity determinants:

  • Evaluate the importance of the C1-A72 mismatch (or equivalent in D. vulgaris)

  • Assess the role of the discriminator base and specific base pairs in the acceptor stem

  • Investigate cross-species activity with initiator tRNAs from different bacteria

Results from E. coli fmt demonstrate the crucial importance of the C1-A72 mismatch, as variants with C1-G72 or G1-C72 base pairs show dramatically reduced formylation efficiency (0.04% and 0.01% of wild-type activity, respectively) . Similar analysis with D. vulgaris fmt would reveal whether the same stringent recognition elements apply or if this enzyme has evolved different substrate preferences.

Table: Relative catalytic efficiencies of E. coli fmt variants with different tRNA substrates

Data adapted from research on E. coli fmt

Which residues in D. vulgaris fmt are critical for tRNA recognition versus catalytic activity?

To distinguish between residues involved in tRNA recognition versus catalytic activity in D. vulgaris fmt, researchers should employ a comprehensive mutational analysis strategy:

Based on studies with E. coli fmt, key residues likely include those equivalent to Arg42 in loop 1, which is critical for tRNA recognition, particularly the C1-A72 mismatch . Mutations in this region significantly alter substrate specificity, as demonstrated by the Δ38-47 deletion variant of E. coli fmt showing 82-fold and 1300-fold increased activity with C1-G72 and G1-C72 tRNA variants, respectively, compared to the wild-type enzyme .

How do mutations in the loop regions of D. vulgaris fmt affect its substrate specificity?

Mutations in loop regions of bacterial formyltransferases can dramatically alter substrate specificity. Based on E. coli fmt studies, loop 1 (residues 34-49) plays a crucial role in recognizing the C1-A72 mismatch in initiator tRNA . A systematic approach to studying the effect of loop mutations in D. vulgaris fmt should include:

• Loop identification and targeting:

  • Identify loops corresponding to E. coli fmt loop 1 through sequence alignment

  • Design deletion variants removing portions of key loops

  • Create point mutations of conserved residues within these loops

• Substrate specificity analysis:

  • Test activity with wild-type initiator tRNA

  • Assess activity with tRNA variants containing different base pairs at positions 1:72

  • Examine cross-species activity with initiator tRNAs from diverse bacteria

• Structural consequences:

  • Analyze structural changes using X-ray crystallography or hydrogen-deuterium exchange mass spectrometry

  • Perform molecular dynamics simulations to understand altered recognition mechanisms

In E. coli fmt, deletion of loop 1 (Δ38-47) fundamentally changes substrate specificity, allowing the enzyme to accommodate tRNA variants with normal base pairing at the top of the acceptor stem . This demonstrates that loop regions can act as specificity determinants rather than just passive binding elements.

How does the function of fmt in D. vulgaris compare with its role in aerobic bacteria?

Desulfovibrio vulgaris is an anaerobic sulfate-reducing bacterium with distinct physiological requirements compared to aerobic bacteria like E. coli. A methodological approach to investigate potential functional differences of fmt in these different environments should include:

• Comparative growth studies:

  • Generate fmt knockout strains of D. vulgaris

  • Compare growth phenotypes with fmt knockouts in aerobic bacteria

  • Perform complementation experiments with fmt genes from different species

• Protein synthesis analysis:

  • Assess in vivo formylation rates under different growth conditions

  • Analyze the effect of environmental factors (pH, temperature, oxidative stress) on fmt activity

  • Examine the relationship between formylation and anaerobic metabolism

• Structural adaptations:

  • Compare thermostability and pH optima of recombinant fmt enzymes from different species

  • Investigate potential redox sensitivity differences between aerobic and anaerobic bacterial formylases

  • Analyze substrate affinity under conditions mimicking the natural environment of each organism

Given the essential role of formylation in bacterial translation initiation, fmt likely serves similar core functions across bacterial species, but may exhibit adaptations to specific environmental niches in terms of stability, regulation, or substrate recognition.

What is the impact of fmt gene knockout or knockdown on D. vulgaris physiology and protein synthesis?

Investigating the impact of fmt manipulation in D. vulgaris requires a comprehensive approach combining genetic, physiological, and biochemical analyses:

• Genetic manipulation strategies:

  • CRISPR-Cas9 gene editing for clean deletion

  • Conditional expression systems (inducible promoters)

  • Antisense RNA or CRISPR interference for knockdown

• Phenotypic characterization:

  • Growth curve analysis under different nutrient conditions

  • Stress tolerance assessment (temperature, pH, oxidative stress)

  • Competitive fitness in mixed cultures

• Protein synthesis analysis:

  • Global proteomics to identify differentially expressed proteins

  • Polysome profiling to assess translation efficiency

  • N-terminal sequencing to confirm formylation status

• Metabolic consequences:

  • Metabolomics to identify pathway alterations

  • Analysis of energy generation systems

  • Examination of folate metabolism and one-carbon transfer reactions

What computational approaches are most effective for modeling the interaction between D. vulgaris fmt and its tRNA substrate?

Computational modeling of D. vulgaris fmt-tRNA interactions requires sophisticated approaches that account for the complex nature of protein-RNA recognition. A comprehensive computational strategy should include:

• Homology modeling:

  • Generate initial models of D. vulgaris fmt based on E. coli fmt crystal structure (PDB ID: 2FMT)

  • Model the initiator tRNA structure of D. vulgaris

  • Dock the tRNA model to the protein model guided by experimental constraints

• Molecular dynamics simulations:

  • Perform all-atom MD simulations of the complex in explicit solvent (100+ ns)

  • Analyze conformational changes in both protein and tRNA

  • Calculate binding free energies using methods like MM-PBSA

• Machine learning approaches:

  • Implement deep learning models trained on protein-RNA interfaces

  • Predict critical interaction points between fmt and tRNA

  • Validate predictions through mutagenesis experiments

• Quantum mechanical calculations:

  • Focus on the active site and catalytic mechanism

  • Model the formyl transfer reaction transition state

  • Calculate energy barriers for catalysis

The computational results should be validated against experimental data whenever possible, particularly through mutagenesis studies testing the importance of predicted interaction residues. This integrated computational-experimental approach provides mechanistic insights that might be challenging to obtain through experimental methods alone.

How do conformational changes in D. vulgaris fmt facilitate catalysis and substrate recognition?

Understanding conformational dynamics in D. vulgaris fmt requires sophisticated structural and biophysical approaches:

• Comparative crystallography:

  • Obtain structures of fmt in multiple states (apo, substrate-bound, product-bound)

  • Analyze structural differences between these states

  • Identify mobile elements involved in catalysis

• Solution-phase dynamics:

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational flexibility

  • Utilize small-angle X-ray scattering (SAXS) to assess solution conformations

  • Apply NMR relaxation experiments to identify mobile regions

• Single-molecule studies:

  • Implement Förster resonance energy transfer (FRET) with strategically placed fluorophores

  • Monitor real-time conformational changes during substrate binding and catalysis

  • Correlate conformational states with catalytic events

• Computational approaches:

  • Perform long-timescale molecular dynamics simulations

  • Implement enhanced sampling techniques like replica exchange

  • Construct Markov state models of the conformational landscape

Based on the E. coli fmt structure, conformational changes likely include movements of the linker and C-terminal domain in response to tRNA binding, and repositioning of key residues like Lys206 to accommodate the 3' terminal adenosine of tRNA . These movements facilitate proper positioning of the methionyl moiety within the hydrophobic cavity of the active site for the formylation reaction.

How has fmt evolved across different bacterial lineages, and what does this reveal about D. vulgaris adaptation?

Evolutionary analysis of fmt across bacterial lineages provides insights into functional adaptation and conservation. A comprehensive evolutionary study should employ:

• Phylogenetic analysis:

  • Collect fmt sequences from diverse bacterial phyla, including both aerobes and anaerobes

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Map key functional residues onto the phylogeny

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Implement branch-site models to detect lineage-specific selection

  • Correlate selection patterns with bacterial ecology and physiology

• Ancestral sequence reconstruction:

  • Infer ancestral fmt sequences at key evolutionary nodes

  • Express and characterize reconstructed ancestral enzymes

  • Compare biochemical properties of ancestral and extant enzymes

• Structural comparison:

  • Map sequence conservation onto structural models

  • Identify structural elements unique to specific bacterial lineages

  • Correlate structural differences with functional specialization

This evolutionary perspective helps contextualize the specific adaptations in D. vulgaris fmt, potentially revealing how this enzyme has been optimized for function in an anaerobic, sulfate-reducing environment compared to aerobic bacteria.

Can recombinant D. vulgaris fmt formylate initiator tRNAs from phylogenetically distant bacteria, and what does this reveal about specificity evolution?

Cross-species activity testing provides valuable insights into the evolution of specificity determinants in fmt enzymes. A systematic methodological approach should include:

• Substrate preparation:

  • Purify initiator tRNAs from diverse bacteria representing different phyla

  • Generate chimeric tRNAs with acceptor stems from one species and body from another

  • Prepare synthetic tRNAs with systematic mutations in key recognition elements

• Activity testing:

  • Measure kinetic parameters (Km, kcat, kcat/Km) with each tRNA substrate

  • Compare relative activities normalized to the cognate tRNA

  • Assess the correlation between activity and phylogenetic distance

• Specificity determinant mapping:

  • Create a matrix of activity versus tRNA sequence features

  • Identify the minimal set of sequence elements required for recognition

  • Test predicted specificity determinants through site-directed mutagenesis

• Structural basis analysis:

  • Model complexes of D. vulgaris fmt with heterologous tRNAs

  • Identify potential steric clashes or missing interactions

  • Validate structural predictions through mutagenesis of both enzyme and tRNA

This cross-species analysis reveals the degree of conservation in the fundamental recognition mechanism of formyltransferases across bacterial evolution, while potentially identifying lineage-specific adaptations in the D. vulgaris enzyme that reflect its ecological niche.