Isolation and characterization of a mutant dihydrofolate reductase-thymidylate synthase from methotrexate-resistant Leishmania cells.

摘要: 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 cloning, expression, characterization of DHFR-TS gene. DNA sequence revealed single base change in position 158 resulted substitution methionine 53 DHFR an arginine. Steady-state measurements purified recombinant enzyme indicated mutation did not cause significant modifications Km or TS substrates but lowered kcat by 4-fold. Of greater interest, there was modification effect on MTX inhibition DHFR. initial complex appeared to been unaffected alteration, subsequent slow-binding step wild-type is absent enzyme. Consequently, overall Ki 30-fold mutant than Transfection L. with gives parasites are capable growing medium containing 10 mM methotrexate, showing itself conferring resistance Leishmania.