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Invariant amino acid replacement affects the dihydrofolate reductase function and its gene expression
1Institute of Protein Research, Academy of Sciences, Moscow Region, USSR.
Protein Engineering
|August 1, 1990
Summary
Mutations in dihydrofolate reductase (DHFR) reveal key roles for specific amino acids. Invariant Thr35 is crucial for protein stability, while Arg57 is essential for DHFR activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Dihydrofolate reductase (DHFR) is a critical enzyme in folate metabolism.
- Understanding DHFR structure-function relationships is vital for drug development, particularly antifolates like trimethoprim (Tmp).
Purpose of the Study:
- To investigate the functional and structural impact of specific amino acid substitutions in Escherichia coli DHFR.
- To elucidate the roles of invariant residues Thr35 and Arg57 in DHFR stability and activity.
Main Methods:
- Oligonucleotide-directed mutagenesis was used to create DHFR variants with amino acid replacements (Thr35Asp, Asn37Ser, Arg57His) and a triple mutant.
- Analysis of mutant protein levels, functional activity, and trimethoprim sensitivity in Escherichia coli.
Main Results:
- The Asn37Ser mutation had no significant effect on DHFR function or cellular levels.
- The Thr35Asp mutation drastically reduced DHFR protein levels, which were partially restored by trimethoprim.
- The Arg57His mutation rendered cells sensitive to trimethoprim, with DHFR levels comparable to wild-type.
Conclusions:
- Invariant residue Thr35 is critical for maintaining a stable DHFR conformation.
- Invariant residue Arg57 is essential for DHFR enzymatic activity.
- Targeted mutagenesis provides insights into DHFR's structural integrity and catalytic function.