Biochemical characterization and essentiality of fumarate hydratase

Abstract

Plasmodium falciparum (Pf), the causative agent of malaria, has an iron-sulfur cluster-containing class I fumarate hydratase (FH) that catalyzes the interconversion of fumarate to malate, a wellknown reaction in the tricarboxylic acid cycle. In humans, the same reaction is catalyzed by class II FH that has no sequence or structural homology with the class I enzyme from Plasmodium. Fumarate is generated in large quantities in the parasite as a byproduct of AMP synthesis and is converted to malate by FH and then used in the generation of the key metabolites oxaloacetate, aspartate, and pyruvate. Previous studies have identified the FH reaction as being essential to P. falciparum, but biochemical characterizations of PfFH that may provide leads for the development of specific inhibitors are lacking. Here, we report on the kinetic characterization of purified recombinant PfFH, functional complementation of fh deficiency in Escherichia coli, and mitochondrial localization in the parasite. We found that the substrate analog mercaptosuccinic acid is a potent PfFH inhibitor, with a K i value in the nanomolar range. The fh gene could not be knocked out in Plasmodium berghei when transfectants were introduced into BALB/c mice; however, fh knockout was successful when C57BL/6 mice were used as host, suggesting that the essentiality of the fh gene to the parasite was mouse strain dependent.Plasmodium falciparum (Pf), the causative agent of the most lethal form of malaria, during its intra-erythrocytic asexual stages, derives ATP primarily from glycolysis with low contribution from mitochondrial pathways (1, 2). The bulk of pyruvate formed is converted to lactic acid with a minor amount entering the tricarboxylic acid (TCA) cycle, the flux through which is upregulated in sexual stages (2). Key intermediates that anaplerotically feed into the TCA cycle are α-ketoglutarate derived from glutamate, oxaloacetate (OAA) from phosphoenolpyruvate, and fumarate from adenosine 5΄-monophosphate (AMP) synthesis. Synthesis of AMP in the parasite is solely from inosine-5΄-monophosphate (IMP) through a pathway involving the enzymes adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL). The net reaction of ADSS and ASL involves consumption of GTP and aspartate and, generation of GDP, P i and fumarate. In the rapidly dividing parasite with an AT-rich genome and high energy requirements, leading to a high demand for adenine pools, one would expect a high flux of fumarate generation. The parasite does not secrete fumarate but instead, the carbon derived from this metabolite can be traced in malate, OAA, aspartate, pyruvate (through PEP) and lactate (3). The metabolic significance of this fumarate anaplerosis is still obscure. In this context, fumarate hydratase (FH, fumarase) the key enzyme to metabolize fumarate becomes an Fumarate hydratase (fumarase, E.C. 4.2.1.2) catalyzes the reversible conversion of fumarate to malate. The stereospecific reaction involves the anti-addition of a water molecule across the carbon-carbon...

Divergent Acyl Carrier Protein Decouples Mitochondrial Fe-S Cluster Biogenesis from Fatty Acid Synthesis in Malaria Parasites

“…Ferredoxin (PF3D7_1214600) is expected to be essential (18), but its main role is to provide electrons for the ISC pathway (39). Class I fumarate hydratase (PF3D7_0927300), which functions in the TCA cycle but may also contribute to purine scavenging, was refractory to disruption in P. falciparum (7) but successfully deleted in P. berghei in a mouse strain-dependent manner (40).…”

“…Ferredoxin (PF3D7_1214600) is expected to be essential (19), but its main role is to provide electrons for the ISC pathway (43). Class I fumarate hydratase (PF3D7_0927300), which functions in the TCA cycle but may also contribute to purine scavenging, was refractory to disruption in P. falciparum (8) but successfully deleted in P. berghei in a mouse strain-dependent manner (44). The Rieske protein (PF3D7_1439400), which is an essential component of ETC complex III (45), is the only known mitochondrial Fe-S cluster protein that has an unequivocally essential function apart from the ISC pathway.…”

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

“…Recently it was shown that 2-thiomalate is also a micromolar inhibitor of class I FHs from parasites Trypanosoma cruzi and Plasmodium falciparum but does not inhibit human FH. 10 , 11 To our knowledge, 2-thiomalate is the first selective small molecule inhibitor of class I FHs, and here we show that this selectivity arises from the binding of the inhibitor to the class I FH catalytic [4Fe-4S] cluster; the human class II FH does not utilize a [4Fe-4S] cluster. In addition, LmFH-1 and LmFH-2 structures show high structural similarity, indicating that inhibitors of one isoform are likely to inhibit the other isoform.…”

“…2-Thiomalate has also been identified as a micromolar competitive inhibitor of class I FHs from Trypanosoma cruzi ( K i, malate = 4.2 ± 0.5 μM for cytosolic isoform; K i, malate = 1.3 ± 0.1 μM for mitochondrial isoform) and Plasmodium falciparum ( K i = 0.32 ± 0.03 μM for S -malate; K i = 0.55 ± 0.05 μM for fumarate) but does not inhibit class II human FH. 10 , 11 To our knowledge, 2-thiomalate is the first selective inhibitor of class I FHs.…”

“… 7 , 8 Class I FHs are [4Fe-4S] cluster-containing dimeric enzymes found in archaea, prokaryotes, and unicellular eukaryotes, including protozoan parasites. 9 − 11 Class II FHs are iron-independent tetrameric enzymes found in prokaryotes and eukaryotes, including humans. 12 Thus, class I FHs are considered attractive drug targets because they are structurally distinct from class II human FH and play vital roles in multiple metabolic pathways.…”