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

• Published in: The Journal of biological chemistry 1994-04, Vol.269 (14), p.10590-10596
• Main Authors: ARREBOLA, R, OLMO, A, RECHE, P, GARVEY, E. P, SANTI, D. V, RUIZ-PEREZ, L. M, GONZALEZ-PACANOWSKA, D
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Cloning, Molecular; Drug Resistance; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Kinetics; Leishmania major; Leishmania major - drug effects; Leishmania major - enzymology; Methotrexate - pharmacology; Mutation; Oxidoreductases; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - isolation & purification; Tetrahydrofolate Dehydrogenase - metabolism; Thymidylate Synthase - genetics; Thymidylate Synthase - isolation & purification; Thymidylate Synthase - metabolism; Transfection; Transferases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)34100-5

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.