Advanced search
Start date
Betweenand
(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

Full text
Author(s):
Feliciano, Patricia R. [1, 2, 3] ; Drennan, Catherine L. [1, 2, 3]
Total Authors: 2
Affiliation:
[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
Abstract

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