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Two glutamyl-tRNA reductase activities in Escherichia coli
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
The Journal of Biological Chemistry
|February 5, 1991
Summary
This study purifies a key enzyme in tetrapyrrole biosynthesis, Glu-tRNA reductase, from E. coli. The enzyme exists in two forms, with the purified monomeric form having a molecular mass of 85 kDa.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Tetrapyrrole biosynthesis is essential for cellular function.
- delta-Aminolevulinic acid (ALA) is the initial precursor in this pathway.
- ALA synthesis in E. coli involves a tRNA-dependent pathway from glutamate, with Glu-tRNA reductase as the rate-limiting enzyme.
Purpose of the Study:
- To purify and characterize the Glu-tRNA reductase enzymes from E. coli K12.
- To investigate the molecular properties and substrate specificity of these enzymes.
Main Methods:
- Enzyme purification using four chromatographic techniques.
- Molecular mass determination via SDS-PAGE, rate zonal sedimentation, and gel filtration.
- Enzyme activity assays in the presence of NADPH.
- Analysis of tRNA substrate specificity.
Main Results:
- Two distinct Glu-tRNA reductase activities were identified with different molecular masses (85 kDa monomer and 45 kDa).
- The purified 85 kDa enzyme is monomeric and active in converting E. coli Glu-tRNA(2Glu) to glutamate 1-semialdehyde.
- Enzyme activity is NADPH-dependent and unaffected by GTP or hemin.
- The enzymes exhibit specific recognition for E. coli Glu-tRNA(2Glu), with poor recognition of homologous tRNAs from other species.
Conclusions:
- E. coli possesses at least two Glu-tRNA reductase activities, likely representing different enzyme forms or isoforms.
- The 85 kDa enzyme is a monomeric Glu-tRNA reductase crucial for ALA biosynthesis.
- The enzyme's tRNA specificity suggests a role in regulating tetrapyrrole synthesis based on E. coli-specific tRNA availability.