FUNCTIONAL EXPRESSION OF THE DIHYDROFOLATE-REDUCTASE DOMAIN OF LEISHMANIA-MAJOR DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE BIFUNCTIONAL PROTEIN

The dihydrofolate reductase (DHFR) domain of the bifunctional dihydrof olate reductase - thymidylate synthase from Leishmania major has been subcloned and expressed as a soluble protein in Escherichia coli strai n PA414 harboring plasmid pLMDHFR. Homogeneous L. major DHFR was obtai ned by chromatography on methotrexate-Sepharose followed by DE52. The purified enzyme migrated as a single 25-kDa protein on SDS-PAGE, The n ative molecular weight was determined to be 26 kDa, indicating that th e isolated domain is a monomer. N-terminal sequence analysis revealed that serine, the second amino acid in the coding sequence, was the N-t erminal amino acid of the protein. The enzyme showed a pH optimum simi lar to that of the bifunctional protein. For purified DHFR, the K-m va lues were <1.0 mu M for H(2)folate and <1.0 mu M for NADPH. The k(cat) of the most active DHFR preparation was 5 s(-1). The K-m and k(cat) v alues were similar to those of the bifunctional enzyme. (C) 1996 Acade mic Press, Inc.