title: Structure and kinetics assays of recombinant Schistosoma mansoni dihydrofolate reductase.
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 recombinant SmDHFR in a heterologous manner to obtain structural, biochemical and kinetic information. X-ray diffraction of recombinant SmDHFR at 1.95Å 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.
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
authors.forename: Vitor Hugo Balasco
authors.initials: VHB
authors.lastname: Serrão
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
authors.forename: Larissa
authors.initials: L
authors.lastname: Romanello
authors.affiliation: National Laboratory of Nanotechnology - LNNano/CNPEM, Campinas-SP, CEP 3083-970, Brazil.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Cassago
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
authors.forename: Juliana Roberta Torini
authors.initials: JRT
authors.lastname: de Souza
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
authors.forename: Juliana
authors.initials: J
authors.lastname: Cheleski
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil.
authors.forename: Ricardo
authors.initials: R
authors.lastname: DeMarco
authors.affiliation: Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK.
authors.forename: José
authors.initials: J
authors.lastname: Brandão-Neto
authors.affiliation: Physics Institute of São Carlos, University of São Paulo, São Carlos-SP, CEP13566-590, Brazil. Electronic address: hmuniz.pereira@gmail.com.
authors.forename: Humberto D'Muniz
authors.initials: HD
authors.lastname: Pereira
mesh_terms: D000818:Animals; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D011994:Recombinant Proteins; D012550:Schistosoma mansoni / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D005493:Folic Acid Antagonists; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate
keywords: Dihydrofolate reductase (DHFR); Isothermal titration calorimetry (ITC); Nucleotide metabolism; Schistosoma mansoni; X-ray crystallography