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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from Leishmania major

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Feliciano, Patricia R. [1, 2, 3] ; Drennan, Catherine L. [1, 2, 3]
Total Authors: 2
[1] MIT, Howard Hughes Med Inst, Cambridge, MA 02139 - USA
[2] MIT, Dept Chem, Cambridge, MA 02139 - USA
[3] MIT, Dept Biol, 77 Massachusetts Ave, Cambridge, MA 02139 - USA
Total Affiliations: 3
Document type: Journal article
Source: BIOCHEMISTRY; v. 58, n. 49, p. 5011-5021, DEC 10 2019.
Web of Science Citations: 0

Class I fumarate hydratases (FHs) are central metabolic enzymes that use a {[}4Fe-4S] cluster to catalyze the reversible conversion of fumarate to S-malate. The parasite Leishmania major, which is responsible for leishmaniasis, expresses two class I FH isoforms: mitochondrial LmFH-1 and cytosolic LmFH-2. In this study, we present kinetic characterizations of both LmFH isoforms, present 13 crystal structures of LmFH-2 variants, and employ site-directed mutagenesis to investigate the enzyme's mechanism. Our kinetic data confirm that both LmFH-1 and LmFH-2 are susceptible to oxygen-dependent inhibition, with data from crystallography and electron paramagnetic resonance spectroscopy showing that oxygen exposure converts an active {[}4Fe-4S] cluster to an inactive {[}3Fe-4S] cluster. Our anaerobically conducted kinetic studies reveal a preference for fumarate over S-malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease k(cat) values 9-16000-fold without substantially affecting K-m values, suggesting that these residues function in catalytic roles. Crystal structures of LmFH-2 variants are consistent with this idea, showing similar bidentate binding to the unique iron of the {[}4Fe-4S] cluster for substrate S-malate as observed in wild type FH. We further present LmFH-2 structures with substrate fumarate and weak inhibitors succinate and malonate bound in the active site and the first structure of an LmFH that is substrate-free and inhibitor-free, the latter showing increased mobility in the C-terminal domain. Collectively, these data provide insight into the molecular basis for the reaction catalyzed by LmFHs, enzymes that are potential drug targets against leishmaniasis. (AU)

FAPESP's process: 14/22246-4 - Mutational analysis of active site residues in fumarate hydratase from Leishmania major
Grantee:Patrícia Rosa Feliciano
Support type: Scholarships abroad - Research Internship - Post-doctor