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Escherichia coli glutamyl-tRNA reductase. Trapping the thioester intermediate
Stefan Schauer1, Shalini Chaturvedi, Lennart Randau
1Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Germany.
The Journal of Biological Chemistry
|October 9, 2002
Summary
Glutamyl-tRNA reductase (GluTR) in E. coli catalyzes a key step in tetrapyrrole biosynthesis. Mutations in the hemA gene often inactivate this enzyme, impacting heme production.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Tetrapyrrole biosynthesis is essential for cellular respiration and photosynthesis.
- Glutamyl-tRNA reductase (GluTR) initiates this pathway in E. coli by converting glutamyl-tRNA to glutamate-1-semialdehyde.
- Understanding GluTR function is crucial for comprehending heme production and related metabolic pathways.
Purpose of the Study:
- To characterize the catalytic mechanism of E. coli glutamyl-tRNA reductase (GluTR).
- To investigate the role of specific amino acid residues in GluTR activity.
- To analyze the impact of mutations on GluTR function and identify key residues for catalysis.
Main Methods:
- Co-expression of hemA and chaperone genes for soluble E. coli GluTR production.
- Mg(2+)-stimulated enzyme assays to detect catalytic intermediates.
- Autoradiography for trapping and detecting thioester intermediates.
- Genetic selection and characterization of hemA mutants.
- Structural analysis based on Methanopyrus kandleri GluTR.
Main Results:
- E. coli GluTR was successfully produced in a soluble, homodimeric form using chaperone co-expression.
- A reactive Cys-50 residue was identified, attacking the tRNA-bound glutamate to form a thioester intermediate.
- NADPH is required for the reduction to glutamate-1-semialdehyde; in its absence, esterase activity was observed.
- Most characterized hemA mutants showed a loss of GluTR activity, with mutations clustering near the catalytic domain.
- A specific mutation (G191D) impaired NADPH binding, resulting in esterase but not reductase activity.
Conclusions:
- The study elucidates the catalytic mechanism of E. coli GluTR, involving a Cys-50 mediated thioester intermediate.
- Mutations affecting key residues, particularly those involved in NADPH binding, abolish reductase activity.
- This work provides insights into the structure-function relationship of GluTR and its role in tetrapyrrole biosynthesis.