Arrebola, R and Olmo, A and Reche, Pedro A and Garvey, Edward P. and Santi, D V and Ruiz Pérez, Luis Miguel and González-Pacanowska, D. (1994) Isolation and characterization of a mutant dihydrofolate reductase-thymidylate synthase from methotrexate-resistant Leishmania cells. The Journal of biological chemistry, 269 (14). pp. 10590-6. ISSN 0021-9258
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.
|Subjects:||Medical sciences > Biology > Biochemistry|
Medical sciences > Biology > Microbiology
Medical sciences > Biology > Molecular biology
|Deposited On:||10 Aug 2009 11:03|
|Last Modified:||01 Sep 2010 12:57|
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