Dataset Information

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

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 kcat values 9-16000-fold without substantially affecting Km 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.

SUBMITTER: Feliciano PR

PROVIDER: S-EPMC7065722 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Feliciano Patricia R PR Drennan Catherine L CL

Biochemistry 20191127 49

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 e ...[more]

PMID: 31743022

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Dataset Information

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

Publications

Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase from <i>Leishmania major</i>.

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