Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain



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Reche, Pedro A and Arrebola, R and Santi, D V and González-Pacanowska, D. and Ruiz Pérez, Luis Miguel (1995) Expression and characterization of the Trypanosoma cruzi dihydrofolate reductase domain. Molecular and Biochemical Parasitology, 76 (1-2). pp. 175-85. ISSN 0166-6851

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We have cloned and expressed in Escherichia coli a 702-base pair gene coding for the dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Trypanosoma cruzi. The DHFR domain was purified to homogeneity by methotrexate-Sepharose chromatography followed by an anion-exchange chromatography step in a mono Q column, and displayed a single 27-kDa band on SDS-PAGE. Gel filtration showed that the catalytic domain was expressed as a monomer. Kinetic parameters were similar to those reported for the wild-type bifunctional enzyme with Km values of 0.75 microM for dihydrofolate and 16 microM for NADPH and a kcat value of 16.5 s-1. T. cruzi DHFR is poorly inhibited by trimethoprim and pyrimethamine and the inhibition constants were always lower for the bifunctional enzyme. The binding of methotrexate was characteristic of a class of inhibitors that form an initial complex which isomerizes slowly to a tighter complex and are referred to as 'slow, tight-binding' inhibitors. While the slow-binding step of inhibition was apparently unaffected in the individually expressed DHFR domain, the overall inhibition constant was two-fold higher as a consequence of the superior inhibition constant value obtained for the initial inhibitory complex.

Item Type:Article
Uncontrolled Keywords:Trypanosoma cruzi; Dihydrofolate reductase; Protozoal enzyme; Heterologous expression; Folate metabolism
Subjects:Medical sciences > Biology > Biochemistry
Medical sciences > Biology > Microbiology
Medical sciences > Biology > Molecular biology
ID Code:9353
Deposited On:14 Aug 2009 10:49
Last Modified:13 Dec 2018 12:48

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