Kinetic and mechanistic study of the enzymatic activity of old yellow enzymes of Leishmania braziliensis and Trypanosoma cruzi

• Grant number: 18/05576-1
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2018
• End date: October 31, 2021
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Julio Cesar Borges
• Grantee: Silvia Helena Libardi
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Oxidoreductase enzymes present a key role in the biochemical process of organisms and thus becoming an important target of parasite control. The Old Yellow Enzyme (OYE), is a NAD(P)H oxidoreductase enzyme which uses one molecule of flavin adenine nucleotide (FMN) as the transient specie to the electron transfer in redox process. The OYE has been identified in fungi, plants and bacteria, but not in metazoans. Pathologies such as visceral leishmaniasis and Chagas disease are induced by protozoan parasites which produce OYE. The chemical mechanism of these enzymes is linked to the electron transfer from NAD(P)H to the FMN which reduces an activated ±, ²-unsaturated activated as ketones, aldehydes, carboxylic acid and derivatives. This project aims to express, purify and functionally characterize the recombinant OYEs from Leishmania braziliensis (LbOYE) and Trypanosoma cruzi (TcOYE). The structure at low and high resolution of TcOYE were characterized by the group and will support part of the studies that will be developed in this proposal, and also the interaction of substrate and inhibitor compounds by protein crystallography. The comparative functionality of these enzymes will be performed using spectrophotometry and Electron Paramagnetic Resonance (EPR) techniques to verify, under different conditions, the electron transfer by the FMN reduction and the radical formation at different substrates and at anaerobic medium. Using these analytical tools will be possible to understand the catalytic mechanism of the enzyme. Then, the basic knowledgment from this project could be used to develop new strategies to the therapy of the disease caused by protozoan such as Chagas and leishmaniosis. This project also will validate the interactions of known lethal compounds to the protozoan with LbOYE and TcOYE. At least two cientific paper in indexed journals in the biochemistry, structural biology and/or molecular biophysics area are intended using the data obtained for this project.