Isolation of cDNAs encoding thymidylate synthase from soybean seedlings and expression of the protein in E. coli.

Abstract

Thymidylate synthase (5,10-methyltetrahydrofolate: dUMP C-methyl-transferase, EC 2.1.1.45) (TS) catalyzes the conversion of deoxyuridylate to thymidylate. Because of its important role in the synthesis of DNA precursors, this enzyme has been extensively studied. The primary structure of thymidylate synthase is highly conserved as revealed by comparison of amino acid sequences of the avian1, mammalian2, and bacterial3 enzymes. Most of these enzymes are homodimers made up of about 35 kDa subunits. However, the enzyme from protozoal parasite, Leishmania major is contained in a bifunctional polypeptide with both thymidylate synthase and dihydrofolate reductase (DHFR) activity. Little is known about TS from plants, although there has been a report of the isolation of a protein complex containing both TS and DHFR activities4. Whether plant TS is a bifunctional enzyme with DHFR or the two enzymes are non-covalently associated can be addressed by cDNA cloning and sequencing. Isolation and expression of the plant TS gene will facilitate characterization of the enzyme at the molecular level, providing biochemical evidence for the study of macromolecule evolution.