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

Biochemistry. 2019 Dec 10;58(49):5011-5021. doi: 10.1021/acs.biochem.9b00923. Epub 2019 Nov 27.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Substitution
  • Catalytic Domain
  • Fumarate Hydratase / chemistry*
  • Fumarate Hydratase / genetics
  • Fumarate Hydratase / metabolism*
  • Iron-Sulfur Proteins / chemistry*
  • Iron-Sulfur Proteins / genetics
  • Iron-Sulfur Proteins / metabolism*
  • Kinetics
  • Leishmania major / chemistry
  • Leishmania major / enzymology*
  • Leishmania major / genetics
  • Multigene Family
  • Oxygen / chemistry
  • Oxygen / metabolism
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*


  • Iron-Sulfur Proteins
  • Protozoan Proteins
  • Fumarate Hydratase
  • Oxygen