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Mutational analysis of active site residues in fumarate hydratase from Leishmania major

Grant number: 14/22246-4
Support Opportunities:Scholarships abroad - Research Internship - Post-doctor
Effective date (Start): February 01, 2015
Effective date (End): January 31, 2016
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal Investigator:Maria Cristina Nonato
Grantee:Patrícia Rosa Feliciano
Supervisor: Catherine L. Drennan
Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Research place: Massachusetts Institute of Technology (MIT), United States  
Associated to the scholarship:13/14988-8 - Elucidation of the mechanism of action of fumarate hydratase from Leishmania major, BP.PD


Leishmaniases, classified as Neglected Tropical Diseases (NTD), are caused by different species of the parasite Leishmania and affect 12 million people worldwide. Fumarate hydratase (FH) catalyzes the stereospecific reversible hydration/dehydration of fumarate to S-malate. Eukaryotes express two isoforms of FH: the mitochondrial isoform (FH-1) that is well known to participate in the tricarboxylic acid (TCA) cycle and may also take part in the succinic fermentation pathway; and the cytosolic isoform (FH-2) that is thought to be involved in the production of fumarate that acts as a substrate for dihydroorotate dehydrogenase, an enzyme involved in the de novo pyrimidine biosynthesis pathway. The vital role of FHs in essential pathways suggests their potential as therapeutic targets. Electron paramagnetic resonance (EPR) approach identified the presence of Fe-S cluster in FHs from Leishmania major (LmFH), and the catalytic efficiency under aerobic and anaerobic environments suggested that these enzymes are highly oxygen sensitive. The crystal structure of the cytosolic FH from L. major (LmFH-2) was solved by SAD techniques using iron as the anomalous scatter. LmFH-2 is the first class I FH structure solved and reveals a previously unknown protein fold. The crystal structure of LmFH-2 was solved in complex with substrate S-malate allowing the identification of the active site in class I FHs. In the present project, we propose to elucidate the catalytic mechanism of LmFHs using site-directed mutagenesis of LmFH-2 active site residues, together with structural and kinetic characterization of the mutants under anaerobic conditions. The results obtained in this project will contribute to the understanding of the class I FH catalytic mechanism as well as will open a new path for fighting leishmaniases. (AU)

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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)
FELICIANO, PATRICIA R.; DRENNAN, CATHERINE L.. Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from Leishmania major. BIOCHEMISTRY, v. 58, n. 49, p. 5011-5021, . (14/22246-4)
FELICIANO, PATRICIA R.; DRENNAN, CATHERINE L.; NONATO, MARIA CRISTINA. Crystal Structures of Fumarate Hydratases from Leishmania major in a Complex with Inhibitor 2-Thiomalate. ACS Chemical Biology, v. 14, n. 2, p. 266-275, . (08/08262-6, 14/22246-4, 13/14988-8)

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