Isolation and Characterization of a Mutant Dihydrofolate Reductase-Thymidylate Synthase from Methotrexate-Resistant Leishmania Cells

Abstract

The MTX-resistant Leishmania major promastigote cell line D7BR1000 displays extrachromosomal amplified R-region DNA, which contains the gene for dihydrofolate reductase-thymidylate synthase (DHFR-TS) (Garvey, E. P., and Santi, D. V. (1986) Science 233, 535-540). Now we report that these methotrexate (MTX)-resistant cells also possessed a structurally altered DHFR-TS. We have performed the cloning, expression, and characterization of the altered DHFR-TS gene. The DNA sequence of the altered DHFR-TS gene revealed a single base change in position 158 which resulted in the substitution of a methionine in position 53 of DHFR for an arginine. Steady-state measurements of the purified recombinant enzyme indicated that the mutation did not cause significant modifications in the Km for DHFR or TS substrates but lowered the kcat by 4-fold. Of greater interest, there was a modification in the effect on MTX inhibition of DHFR. The initial inhibition complex appeared to have been unaffected by the alteration, but the subsequent slow-binding step of inhibition in the wild-type enzyme is absent in the altered enzyme. Consequently, the overall Ki for MTX was 30-fold greater for the mutant than for the wild-type enzyme. Transfection of L. major with the mutant DHFR-TS gene gives parasites that are capable of growing in medium containing 10 mM methotrexate, showing that the altered DHFR gene is in itself capable of conferring MTX resistance in Leishmania.