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Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase

DOI: 10.1016/j.actatropica.2017.03.007 DOI Help

Authors: Vitor Hugo Balasco Serrao (University of São Paulo) , Larissa Romanello (University of São Paulo) , Alexandre Cassago (National Laboratory of Nanotechnology –LNNano/CNPEM) , Juliana Roberta Torini De Souza (University of São Paulo) , Juliana Cheleski (University of São Paulo) , Ricardo Demarco (University of São Paulo) , Jose Brandao-neto (Diamond Light Source) , Humberto D'muniz Pereira (University of São Paulo)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Tropica

State: Published (Approved)
Published: March 2017

Abstract: The parasite Schistosoma mansoni possesses all pathways for pyrimidine biosynthesis, in which dihydrofolate reductase (DHFR), thymidylate cycle participants, is essential for nucleotide metabolism to obtain energy and structural nucleic acids. Thus, DHFRs have been widely suggested as therapeutic targets for the treatment of infectious diseases. In this study, we expressed recombinantSmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinantSmDHFR at 1.95 Å resolution showed that the structure exhibited the canonical DHFR fold. Isothermal titration calorimetry was used to determine the kinetic constants for NADP+ and dihydrofolate. Moreover, inhibition assays were performed using the commercial folate analogs methotrexate and aminopterin; these analogs are recognized as folate competitors and are used as chemotherapeutic agents in cancer and autoimmune diseases. This study provides information that may prove useful for the future discovery of novel drugs and for understanding these metabolic steps from this pathway of S. mansoni, thus aiding in our understanding of the function of these essential pathways for parasite metabolism.

Journal Keywords: Schistosoma mansoni; Nucleotide metabolism; Dihydrofolate reductase (DHFR); Isothermal titration calorimetry (ITC); X-ray crystallography

Subject Areas: Biology and Bio-materials, Chemistry

Facility: ESRF