6UPO

Crystal structure of H334A variant of cytosolic fumarate hydratase from Leishmania major in a complex with S-malate

Summary for 6UPO

• Entry DOI: 10.2210/pdb6upo/pdb
• Related: 6UNZ 6UO0 6UOI 6UOJ 6UP9 6UPM 6UQ8 6UQ9 6UQB 6UQL 6UQM 6UQN
• Descriptor: Fumarate hydratase 2, IRON/SULFUR CLUSTER, (2S)-2-hydroxybutanedioic acid, ... (4 entities in total)
• Functional Keywords: fumarate hydratase, fumarase, leishmania major, substrate, variant h334a, s-malate, lyase
• Biological source: Leishmania major strain Friedlin
• Total number of polymer chains: 2
• Total formula weight: 133864.25

Authors

Feliciano, P.R.,Drennan, C.L. (deposition date: 2019-10-18, release date: 2019-12-04, Last modification date: 2023-10-11)

Primary citation

Feliciano, P.R.,Drennan, C.L.
Structural and Biochemical Investigations of the [4Fe-4S] Cluster-Containing Fumarate Hydratase fromLeishmania major.
Biochemistry, 58:5011-5021, 2019

PubMed Abstract: Class I fumarate hydratases (FHs) are central metabolic enzymes that use a [4Fe-4S] cluster to catalyze the reversible conversion of fumarate to -malate. The parasite , 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 -malate. Our data further reveal that single alanine substitutions of T467, R421, R471, D135, and H334 decrease values 9-16000-fold without substantially affecting 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 -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.

PubMed: 31743022
DOI: 10.1021/acs.biochem.9b00923

Experimental method

X-RAY DIFFRACTION (3.113 Å)