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Conversion of thymidylate synthase into an HIV protease substrate
J J Kupiec1, S Hazebrouck, T Leste-Lasserre
1Génétique des Virus, ICGM-CNRS UPR 415, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France.
The Journal of Biological Chemistry
|August 2, 1996
Summary
Researchers engineered thymidylate synthase (TS) to be cleaved by HIV-1 protease. This enzyme modification controlled bacterial growth, offering a new method for selecting protease inhibitors.
Area of Science:
- Molecular Biology
- Enzymology
- Genetics
Background:
- Thymidylate synthase (TS) is a critical enzyme in DNA synthesis.
- Understanding TS function is key to developing targeted therapies.
Purpose of the Study:
- To investigate insertional mutagenesis of the Escherichia coli TS gene (thyA).
- To engineer TS as a substrate for Human immunodeficiency virus type 1 (HIV-1) protease.
- To explore protease-mediated control of essential metabolic enzymes.
Main Methods:
- Extensive insertional mutagenesis of the E. coli thyA gene using three distinct methods.
- Identification of TS sequence zones permissive for exogenous sequence insertion.
- Insertion of HIV-1 protease substrate sequences into permissive TS sites.
Main Results:
- Mutants with defective TS were generated through random insertions.
- Specific permissive sites in TS were identified, allowing for functional insertions.
- Engineered TS was cleaved by HIV-1 protease in vivo, leading to a thymidylate synthase-deficient phenotype in some E. coli strains.
- Permissive sites were confirmed to be non-essential for enzyme activity and accessible to HIV protease.
Conclusions:
- Protease-mediated cleavage of essential enzymes can control cellular phenotypes.
- These engineered TS mutants are valuable tools for genetic selection of protease inhibitors and for analyzing protease specificities.