Abstract | INTRODUCTION: MATERIALS AND METHODS: The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. RESULTS: The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed Km and Vmax values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H₂F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity (Ki = 22.0 µM) than trimethoprim (Ki = 33 µM) and pyrimethamine (Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. CONCLUSION: The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.
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Authors | Edison Osorio, Carolina Aguilera, Nelson Naranjo, Marcel Marín, Carlos Muskus |
Journal | Biomedica : revista del Instituto Nacional de Salud
(Biomedica)
2013 Jul-Sep
Vol. 33
Issue 3
Pg. 393-401
ISSN: 2590-7379 [Electronic] Colombia |
PMID | 24652175
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Chemical References |
- Folic Acid Antagonists
- Multienzyme Complexes
- thymidylate synthase-dihydrofolate reductase
- Tetrahydrofolate Dehydrogenase
- Thymidylate Synthase
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Topics |
- Folic Acid Antagonists
(pharmacology)
- Leishmania
(enzymology)
- Multienzyme Complexes
(antagonists & inhibitors, chemistry)
- Tetrahydrofolate Dehydrogenase
(chemistry)
- Thymidylate Synthase
(antagonists & inhibitors, chemistry)
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