Isolation and characterization of a mutant dihydrofolate reductase-thymidylate synthase from methotrexate-resistant Leishmania cells.
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.
Arrebola
R
R
Instituto de Parasitología y Biomedicina Lopez-Neyra, Consejo Superior de Investigaciones Cientificas, Granada, Spain.
Olmo
A
A
Reche
P
P
Garvey
E P
EP
Santi
D V
DV
Ruiz-Perez
L M
LM
Gonzalez-Pacanowska
D
D
D000818:Animals; D003001:Cloning, Molecular; D004351:Drug Resistance; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D018320:Leishmania major / Q000187:drug effects / Q000201:enzymology*; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D008727:Methotrexate