Crystal Structures of Fumarate Hydratases from Leishmania major in a Complex with Inhibitor 2-Thiomalate

Abstract

Leishmaniases affect the poorest people on earth and have no effective drug therapy. Here, we present the crystal structure of the mitochondrial isoform of class I fumarate hydratase (FH) from Leishmania major and compare it to the previously determined cytosolic Leishmania major isoform. We further describe the mechanism of action of the first class-specific FH inhibitor, 2-thiomalate, through X-ray crystallography and inhibition assays. Our crystal structures of both FH isoforms with inhibitor bound at 2.05 Å resolution and 1.60 Å resolution show high structural similarity. These structures further reveal that the selectivity of 2-thiomalate for class I FHs is due to direct coordination of the inhibitor to the unique Fe of the catalytic [4Fe-4S] cluster that is found in class I parasitic FHs but is absent from class II human FH. These studies provide the structural scaffold in order to exploit class I FHs as potential drug targets against leishmaniases as well as Chagas diseases, sleeping sickness, and malaria.

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

“…Recently, we determined the crystal structures of mitochondrial and cytosolic isoforms of class I FHs in L. major , a parasite responsible for the neglected tropical disease leishmaniasis. 5 , 14 These structures revealed that mitochondrial LmFH-1 and cytosolic LmFH-2 have very similar homodimeric structures with each monomer composed of two domains, an N-terminal domain and a C-terminal domain, arranged around the catalytic [4Fe-4S] cluster. 5 , 14 A co-crystal structure of LmFH-2 revealed substrate S -malate bound to the unique iron of the [4Fe-4S] cluster as well as conserved active site residues that play a key role in catalysis.…”

“… 5 , 14 These structures revealed that mitochondrial LmFH-1 and cytosolic LmFH-2 have very similar homodimeric structures with each monomer composed of two domains, an N-terminal domain and a C-terminal domain, arranged around the catalytic [4Fe-4S] cluster. 5 , 14 A co-crystal structure of LmFH-2 revealed substrate S -malate bound to the unique iron of the [4Fe-4S] cluster as well as conserved active site residues that play a key role in catalysis. 5 In this study, we present mutagenesis, biochemical, and structural characterization of LmFH-2 and propose a catalytic mechanism for class I FHs.…”

“…Interestingly, malonate does not coordinate the cluster in contrast to the inhibitors succinate (this work) and 2-thiomalate. 14 These observations indicate that inhibitors can bind to LmFH in multiple ways to afford inactivation, albeit some of the inhibition measured is quite modest.…”

“…Recently, we found that LmFH isoforms have very similar homodimeric structures, with each monomer comprised of an N-terminal domain and a C-terminal domain, connected by a flexible linker, that are arranged around the catalytic [4Fe-4S] cluster. 5 , 14 An LmFH-2 co-crystal structure revealed substrate S -malate bound in a bidentate fashion to the [4Fe-4S] cluster in the active site as well as at a cavity located between the N- and C-terminal domains that leads to the active site. 5 This structure suggested a key catalytic role for the conserved active site residues T467, R421, R471, D135, H334, and R173, as well as C-terminal domain mobility that could regulate access of the substrate to the active site.…”

<em>Leishmania</em> Proteomics: An <em>in Silico</em> Perspective

“…109 6MSO catalyzes the stereospecific reversible conversion of fumarate to S-malate. This reaction is part of the tricarboxylic acid (TCA) cycle, takes part of the succinic fermentation pathway, participates in DNA repair processes and is proposed to provide fumarate for the de novo pyrimidine biosynthetic pathway 110. Finally, aldolase L. Peptidyl-prolyl cis-trans isomerase (PDB ID: 4S1E) accelerates the folding process of proteins.…”