| Texto completo | |
| Autor(es): |
Feliciano, Patricia R.
;
Drennan, Catherine L.
Número total de Autores: 2
|
| Tipo de documento: | Artigo Científico |
| Fonte: | BIOCHEMISTRY; v. 58, n. 49, p. 11-pg., 2019-12-10. |
| Resumo | |
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) | |
| Processo FAPESP: | 14/22246-4 - Análise mutacional dos resíduos do sítio ativo de fumarato hidratase de Leishmania major |
| Beneficiário: | Patrícia Rosa Feliciano |
| Modalidade de apoio: | Bolsas no Exterior - Estágio de Pesquisa - Pós-Doutorado |