Kinetic and structural studies of the Deoxycytidylate deaminase (DEOC) from Schistosoma mansoni

• Grant number: 12/15777-8
• Support type: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): November 01, 2012
• Effective date (End): February 28, 2014
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal researcher: Humberto D'Muniz Pereira
• Grantee: Jessica Fernandes Scortecci
• Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Schistosomiasis is a neglected disease that doesn't attract large investments of the pharmaceutical companies in the search and development for new potential drugs. The enzyme Deoxycytidylate deaminase (DEOC) is one of the enzymes that belongs to the thymidylate cycle of the parasite Schistosoma mansoni. The thymidylate cycle is an essential metabolic pathway of the parasite and the enzyme DEOC acts in the irreversible hydrolytic deamination of deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate (dUMP) to provide the substrate for thymidylate synthase, an essential step in the synthesis of structural nucleotides (DNA and RNA) and energy (UTP). This study aims to determine a kinetic model allowing the search for inhibitors and also get the structural model which makes possible the understanding of site-specific interactions contributing to the increase of knowledge about the fundamental ways of the parasite. Will be used the technique of heterologous expression in Escherichia coli BL21 (DE3) system of the SmDEOC clone in pET28a vector (Novagen), purification by affinity chromatography and molecular exclusion after cleavage of the His-TAG with thrombin, validation by SDS-PAGE and native electrophoresis. The kinetic assays will be done by using fluorimetric and monitoring the decrease of absorption at 290 nm (wavelength related to dCMP). The model structure will be obtained by using X-ray diffraction in monocrystal shaped by vapor diffusion method in hanging drop. The cDNA of SmDEOC was amplified, the recombinant enzyme was expressed and characterized as a stable hexamer in solution (118 kDa) by chromatographic profile analysis and electrophoresis in the default of SDS and denaturing conditions. The first crystallization trials already indicate the favorable conditions obtaining a few crystals to be tested. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SCORTECCI, JESSICA FERNANDES; BALASCO SERRAO, VITOR HUGO; CHELESKI, JULIANA; TORINI, JULIANA ROBERTA; ROMANELLO, LARISSA; DEMARCO, RICARDO; PEREIRA, HUMBERTO D'MUNIZ. Spectroscopic and calorimetric assays reveal dependence on dCTP and two metals (Zn2+ + Mg2+) for enzymatic activity of Schistosoma mansoni deoxycytidylate (dCMP) deaminase. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1865, n. 11, A, p. 1326-1335, NOV 2017. Web of Science Citations: 1.