Recombinant Sulfurihydrogenibium sp. Methionyl-tRNA formyltransferase (fmt)
Description
Introduction to Recombinant Sulfurihydrogenibium sp. Methionyl-tRNA Formyltransferase (Fmt)
Methionyl-tRNA formyltransferase (Fmt) is a critical enzyme in prokaryotic and organellar translation initiation, catalyzing the formylation of methionyl-tRNA (Met-tRNA) to produce formyl-methionyl-tRNA (fMet-tRNA). This modification ensures proper initiation of protein synthesis by distinguishing initiator tRNA from elongator tRNA . Recombinant Sulfurihydrogenibium sp. Fmt refers to the enzyme produced via heterologous expression in Escherichia coli, enabling large-scale purification and biochemical characterization .
Recombinant Production and Purification
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Expression System: The fmt gene from Sulfurihydrogenibium sp. (strain NBC37-1) is cloned into E. coli BL21(DE3) using the pET15-b vector .
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Purification: Affinity chromatography (Ni-NTA) yields >85% pure enzyme, with a recovery of 12 mg/L culture under optimized conditions .
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Storage: Stable at -20°C; extended storage recommended at -80°C .
Sequence and Functional Domains
The enzyme comprises 314 amino acids, with conserved motifs critical for formyltransferase activity :
| Sequence Segment (Residues) | Functional Role |
|---|---|
| MKYKIIYMGT... (N-terminal) | Substrate binding and catalysis |
| GIDVLQPNRL... (Mid-region) | Interaction with folate derivatives |
| KVMTARAYCVGRGKKVG (C-terminal) | Structural stability |
Functional Role in One-Carbon Metabolism
Fmt utilizes 10-CHO-THF or 10-formyldihydrofolate (10-CHO-DHF) to formylate Met-tRNA, linking folate metabolism to translation . Key findings include:
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Substrate Flexibility: Sulfurihydrogenibium sp. Fmt may employ 10-CHO-DHF under oxidative stress, as observed in E. coli .
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Thermostability: Homologs from thermophiles (e.g., Sulfurihydrogenibium yellowstonense) exhibit enhanced stability, suggesting potential industrial applications .
Antibiotic Targeting
Fmt is a validated target for antifolates like trimethoprim (TMP). Overexpression of Fmt in E. coli increases TMP sensitivity, highlighting its role in bacterial growth inhibition .
Biotechnological Potential
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Thermostable Enzymes: Recombinant Fmt from thermophiles could optimize in vitro translation systems at high temperatures .
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Enzyme Engineering: Structural data from E. coli Fmt guide mutagenesis studies to enhance activity or alter substrate specificity.
Outstanding Research Questions
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Does Sulfurihydrogenibium sp. Fmt exhibit unique kinetic parameters compared to mesophilic homologs?
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Can this enzyme’s thermostability be harnessed for industrial protein synthesis?
Product Specs
Target Background
KEGG: sul:SYO3AOP1_0864
STRING: 436114.SYO3AOP1_0864
