Recombinant Erwinia tasmaniensis Methionyl-tRNA formyltransferase (fmt)
Description
Functional Role of Fmt
Fmt transfers a formyl group from 10-formyltetrahydrofolate (10-CHO-THF) or 10-formyldihydrofolate (10-CHO-DHF) to methionine charged on tRNA<sup>Met</sup>, ensuring accurate translation initiation. In E. tasmaniensis, this enzyme likely supports the bacterium’s metabolic adaptability in plant-associated environments, where efficient protein synthesis is critical for survival and competition . Notably, E. tasmaniensis lacks pathogenic traits but shares genomic features with phytopathogenic Erwinia species, suggesting conserved translational machinery .
Genomic Context in Erwinia tasmaniensis
The E. tasmaniensis Et1/99 genome (RefSeq: GCF_000026185.1) includes a chromosomal fmt gene, part of its core translation machinery. Key genomic insights include:
Biochemical Characterization
While direct studies on recombinant E. tasmaniensis Fmt are absent, inferences can be drawn from related enzymes:
Catalytic Activity
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Substrate Specificity: E. coli Fmt utilizes 10-CHO-THF and 10-CHO-DHF as formyl donors . Mutational studies in E. coli show that substitutions like A89L (analogous to human S125L) reduce activity by >100-fold .
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Kinetic Parameters:
Recombinant Expression and Applications
Recombinant Fmt production typically involves cloning the fmt gene into E. coli expression systems. Key considerations include:
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Inducible Expression: Use of T7 or arabinose promoters for controlled enzyme production .
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Purification: Affinity chromatography (e.g., His-tag) followed by gel filtration .
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Biotechnological Relevance:
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Antifolate Drug Studies: Fmt’s reliance on folate derivatives makes it a target for antifolate therapeutics. E. coli Fmt sensitivity to trimethoprim highlights its role in one-carbon metabolism .
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Synthetic Biology: Engineered fmt variants could modulate translation fidelity in recombinant protein production systems .
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Research Gaps and Future Directions
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Enzyme Kinetics: Direct measurement of E. tasmaniensis Fmt activity with 10-CHO-THF/10-CHO-DHF is needed.
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Structural Analysis: X-ray crystallography or cryo-EM could elucidate substrate-binding nuances.
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Pathway Integration: How Fmt interacts with E. tasmaniensis’s unique metabolic pathways (e.g., levan synthesis ) remains unexplored.
Product Specs
Target Background
KEGG: eta:ETA_31290
STRING: 465817.ETA_31290
Q&A
What is the biological function of Methionyl-tRNA formyltransferase in bacteria?
Methionyl-tRNA formyltransferase (fmt) catalyzes the formylation of initiator methionyl-tRNA (Met-tRNA^Met) to formylmethionyl-tRNA (fMet-tRNA^Met), which is essential for translation initiation in bacteria, mitochondria, and chloroplasts. This enzyme transfers a formyl group from 10-formyl-tetrahydrofolate (10-CHO-THF) to the amino group of the methionine moiety attached to the initiator tRNA. The resulting formylated initiator tRNA serves as a substrate for initiation factor IF2 and acts as a negative determinant for elongation factor EF-Tu, ensuring proper discrimination between initiation and elongation processes in protein synthesis . In Erwinia tasmaniensis, like other bacteria, this formylation step marks the beginning of protein synthesis and is crucial for efficient translation initiation.
What expression systems are recommended for producing recombinant E. tasmaniensis fmt?
Based on successful expression strategies for other fmt proteins, the following approach is recommended:
The expression protocol should be optimized by testing various induction times, temperatures, and media compositions to achieve maximum yield of soluble, active enzyme.
How do mutations in conserved residues affect E. tasmaniensis fmt catalytic activity?
Studies on human mitochondrial MTF have shown that mutations of conserved serine residues significantly impact enzyme activity. Specifically, the S125L mutation reduced activity by 653-fold, while the S209L mutation decreased activity by 36-fold . Corresponding mutations in E. coli MTF (A89L and A172L, respectively) showed similar effects, with activity reductions of 144-fold and 4-fold . These findings suggest that strategic positioning of small aliphatic amino acids is critical for normal fmt function. For E. tasmaniensis fmt, site-directed mutagenesis targeting homologous residues would likely produce comparable effects on enzyme activity.
What is the mechanism of formyl transfer and how can it be studied in E. tasmaniensis fmt?
The chemical mechanism of formyl transfer by fmt enzymes involves:
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Binding of 10-CHO-THF or alternative formyl donor
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Binding of methionyl-tRNA^Met
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Nucleophilic attack by the α-amino group of methionine on the formyl carbon
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Release of products (fMet-tRNA^Met and THF or DHF)
To investigate this mechanism in E. tasmaniensis fmt, researchers should employ:
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Pre-steady-state kinetics to identify rate-limiting steps
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Isotope labeling with ^13C or ^14C formyl groups to track transfer
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X-ray crystallography of enzyme-substrate complexes
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Site-directed mutagenesis of putative catalytic residues
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pH-rate profiles to identify critical ionizable groups
These approaches would provide insights into transition states and the roles of specific residues in catalysis.
What is the optimal purification strategy for obtaining active recombinant E. tasmaniensis fmt?
Based on successful purification protocols for related fmt proteins, the following multistep procedure is recommended:
| Purification Step | Conditions | Purpose |
|---|---|---|
| Cell lysis | Sonication or French press in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT | Release protein while maintaining stability |
| Affinity chromatography | Ni-NTA column, imidazole gradient elution | Capture His-tagged protein |
| Ion exchange | Resource Q column, pH 8.0, NaCl gradient | Remove remaining contaminants |
| Size exclusion | Superdex 75 column, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM DTT | Final polishing, buffer exchange |
| Storage | Add glycerol to 50%, flash freeze in liquid nitrogen | Maintain long-term activity |
Throughout purification, it is critical to monitor enzyme activity using the appropriate assay to ensure retention of catalytic function.
How can the activity of recombinant E. tasmaniensis fmt be measured in vitro?
Multiple complementary assays can be employed to assess fmt activity:
For kinetic characterization, the assay should be optimized to determine Km and Vmax values for both tRNA and folate substrates under various conditions.
What approaches can be used to investigate the structure-function relationship in E. tasmaniensis fmt?
A comprehensive structure-function analysis would combine:
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Homology modeling based on known fmt structures
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Systematic site-directed mutagenesis targeting:
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Conserved residues identified through sequence alignment
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Predicted substrate-binding sites
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Catalytic residues
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Kinetic analysis of mutant proteins to determine:
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Effects on Km for each substrate
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Changes in Vmax and catalytic efficiency (Vmax/Km)
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Alterations in substrate specificity
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Structural studies using X-ray crystallography or cryo-EM
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Molecular dynamics simulations to understand conformational changes during catalysis
How can knowledge of E. tasmaniensis fmt contribute to understanding antifolate resistance mechanisms?
Research on E. coli fmt has shown that fmt overexpression increases sensitivity to trimethoprim (TMP), an antifolate drug . This suggests a complex relationship between fmt activity and folate metabolism that could inform studies on antifolate resistance mechanisms. For E. tasmaniensis fmt, investigation of this relationship would involve:
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Creating fmt overexpression and deletion strains
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Testing sensitivity to various antifolate compounds
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Measuring intracellular levels of folate derivatives under antifolate stress
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Analyzing changes in fmt activity with different folate pools
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Comparing responses with those of pathogenic species
This research could provide insights into how non-pathogenic bacteria like E. tasmaniensis maintain protein synthesis initiation under folate-limited conditions, potentially revealing novel resistance mechanisms.
What role does E. tasmaniensis fmt play in bacterial adaptation to environmental stress?
As a non-phytopathogenic bacterium found on apple and pear trees , E. tasmaniensis faces various environmental stresses including temperature fluctuations, UV exposure, and nutrient limitations. Investigation of fmt's role in stress adaptation would include:
| Stress Condition | Experimental Approach | Expected Outcomes |
|---|---|---|
| Temperature stress | Growth analysis of wildtype vs. fmt mutants at various temperatures | Determine if fmt activity is critical at temperature extremes |
| Nutrient limitation | Measure fmt expression and activity under folate-limiting conditions | Identify regulatory mechanisms for maintaining translation |
| Oxidative stress | Expose cultures to oxidative agents and monitor fmt activity | Assess sensitivity of fmt to oxidation and potential compensatory mechanisms |
| pH stress | Compare protein synthesis rates at different pH levels | Determine optimal pH range for fmt function in vivo |
These studies would reveal how E. tasmaniensis balances protein synthesis initiation requirements with environmental constraints.
How does E. tasmaniensis fmt function compare with mitochondrial MTF in terms of disease-related mutations?
Studies on human mitochondrial MTF have identified mutations associated with Leigh syndrome, a severe neurological disorder . Comparative analysis of E. tasmaniensis fmt and human mitochondrial MTF could provide insights into the molecular basis of disease-causing mutations:
Creating equivalent mutations in E. tasmaniensis fmt and characterizing their biochemical properties could provide a bacterial model system for studying mitochondrial disease mechanisms, potentially revealing evolutionary conservation of critical functional residues.
What methodological advances could enhance studies of E. tasmaniensis fmt?
Several emerging technologies could significantly advance research on E. tasmaniensis fmt:
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CRISPR-Cas9 genome editing for precise chromosomal manipulation
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Single-molecule enzymology to observe individual catalytic events
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Cryo-EM for structural determination without crystallization
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Time-resolved X-ray crystallography to capture reaction intermediates
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Nanopore sequencing to monitor tRNA modifications directly
Implementation of these techniques would provide unprecedented insights into fmt function at molecular and cellular levels.
How might E. tasmaniensis fmt research contribute to understanding non-canonical functions of formylation?
Beyond its canonical role in translation initiation, fmt-mediated formylation may serve additional functions:
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Potential roles in stress signaling pathways
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Interactions with bacterial immune evasion mechanisms
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Contributions to biofilm formation
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Involvement in bacterial communication systems
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Impact on codon usage and translational regulation
Comparative studies between pathogenic bacteria and non-pathogenic E. tasmaniensis could reveal whether formylation has evolved specialized functions in different ecological niches.
What is the evolutionary significance of fmt conservation in non-pathogenic bacteria?
As a non-phytopathogenic bacterium , E. tasmaniensis represents an interesting subject for evolutionary studies of fmt conservation. Research questions include:
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Is fmt under different selective pressure in non-pathogenic versus pathogenic bacteria?
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Has horizontal gene transfer influenced fmt evolution in Erwinia species?
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Do environmental conditions in its ecological niche (apple and pear trees) drive specific adaptations in fmt function?
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How does fmt sequence conservation compare with other translation factors across Erwinia species?
Such evolutionary analyses would provide context for understanding the fundamental importance of formylation in bacterial protein synthesis across diverse ecological niches.
