title: Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania (Viannia) and its evaluation as a drug target.
abstract: BACKGROUND
  Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania (Viannia) species.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Grupo de Investigación en Sustancias Bioactivas, GISB, Facultad de Química Farmacéutica, Universidad de Antioquia.
authors.forename: Edison
authors.initials: E
authors.lastname: Osorio
authors.affiliation: Biología de la Conservación y Biotecnología, Facultad de Ciencias Básicas, Corporación Universitaria Santa Rosa de Cabal, Santa Rosa de Cabal, Colombia.
authors.forename: Carolina
authors.initials: C
authors.lastname: Aguilera
authors.affiliation: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Universidad de Antioquia, Medellín, Colombia.
authors.forename: Nelson
authors.initials: N
authors.lastname: Naranjo
authors.affiliation: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Universidad de Antioquia, Medellín, Colombia.
authors.forename: Marcel
authors.initials: M
authors.lastname: Marín
authors.affiliation: Programa de Estudio y Control de Enfermedades Tropicales, PECET, Universidad de Antioquia, Medellín, Colombia.
authors.forename: Carlos
authors.initials: C
authors.lastname: Muskus
mesh_terms: D005493:Folic Acid Antagonists / Q000494:pharmacology*; D007891:Leishmania / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase