Structural and biochemical characterization of Schistosoma mansoni class II fumarate hydratase enzyme

• Autor(a) principal: Cardoso, Iara Aimê
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Biblioteca Digital de Teses e Dissertações da USP
• Texto Completo: http://www.teses.usp.br/teses/disponiveis/60/60136/tde-28032020-215307/
• topic: Caracterização cinética; Cristalografia de raios-x; Fumarate hydratase; Fumarato hidratase; Kinetic characterization; Schistosoma mansoni; Schistosoma mansoni; X-ray crystallography

Schistomiasis is a neglected tropical disease caused by trematodes worms from the genus Schistosoma. Schistosomiasis is the second most devastating parasitic disease after malaria. The disease has a high economic burden and affects mainly poor population without access to proper sanitation. Praziquantel is the only drug approved for the treatment of schistosomiasis and resistance is already reported. Fumarate hydratases or fumarases are enzymes that catalyze the reversible hydration of fumarate to L-malate. This enzyme participates in DNA repair and important metabolic processes such as the urea and the tricarboxylic acid cycles. Fumarases are divided in two classes, and Schistosoma mansoni possess both, being class I localized in mitochondria, while class II is cytosolic. The fundamental role of fumarases in the metabolism make them potential target for drug design against schistosomiasis. This work describes, for the first time, the cloning, expression and purification protocol for the class II fumarate hydratase from Schistosoma mansoni (SmFHII). In order to estimate the contribution of the reverse reaction, the enzyme was kinetically characterized using both substrates concomitantly. SmFHII was shown to follow a MichaelisMenten mechanism of catalysis with of 19 mM-1s-1 and of 49 mM-1s-1, and of 0.56 mM and of 0.15 mM. Differential scanning fluorimetry (DSF) performed under different chemical environments shows that the highest thermal stability is reached at pH 7.5 and at higher ionic strength. The significant thermoshift observed for SmFHII in presence of well known ligands makes DSF the adequate technique for ligand screening. SmFHII structure in complex with L-malate was determined by single crystal X-ray diffraction, at 1.85 Å resolution. A new construct [SmFHII(Δ263-277)] lacking the additional portion only found in trematode worms was also evaluated by kinetic and DSF experiments. Although not essential for activity, the results suggest that the removal of this region impacts on protein stability and may has influence on L-malate catalysis. The differences between SmFHII and human fumarase are distributed all over the structure, and could be explored to design new selective inhibitors.