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

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

Patricia R Feliciano  1   2   3 , Catherine L Drennan  1   2   3
Affiliations

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

Patricia R Feliciano et al. Biochemistry. .

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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Scheme 1
Scheme 1. FH Reversible Reaction
Figure 1
Figure 1
Initial rates of LmFH isoforms and LmFH-2 variants as a function of S-malate and fumarate concentration. (A) Initial velocities of LmFH isoforms vs S-malate concentration. (B) Initial velocities of LmFH isoforms vs fumarate concentration. (C) Initial velocities of LmFH-2 variants vs S-malate concentration. (D) Initial velocities of LmFH-2 variants vs fumarate concentration. The plots were fit with the Michaelis–Menten equation (eq 1). The Y-axis on the right corresponds to the initial velocities of the LmFH-2-H334A variant, which are colored gray. The error bars were calculated from triplicate reactions. Error bars are given for all measurements but are not easily visible in all cases. (E) Catalytic efficiency (kcat/Km) of LmFH isoforms and LmFH-2 variants for S-malate and fumarate.
Figure 2
Figure 2
Active sites of LmFH-2 variants reveal similar S-malate-binding modes with LmFH-2-R173A displaying the largest variation. (A) LmFH-2 in a complex with S-malate. The proposed catalytic residues are colored yellow (D135 and H334 from chain B) and light blue (T467, R421, and R471). Residue R173 that is proposed to play a role in the correct positioning of the substrate in the active site is colored magenta. The distance from the T467 OH group to the S-malate C3 is shown as a green dashed line. (B) LmFH-2-H334A in a complex with S-malate. The H334A substitution is colored yellow. (C) LmFH-2-R421A in a complex with S-malate. The R421A substitution is colored light blue. (D) LmFH-2-T467A in a complex with S-malate. The T467A substitution is colored light blue. (E) LmFH-2-R471A in a complex with S-malate. The R471A substitution is colored light blue. (F) LmFH-2-R173A in a complex with S-malate. The R173A substitution is colored magenta. The substrate S-malate is colored green. The [4Fe-4S] cluster is shown as orange (Fe) and yellow (S) spheres. The water molecules are shown as red spheres. The hydrogen bonds are shown as gray dashed lines.
Figure 3
Figure 3
Structures of LmFH-2 variants bound to succinate or fumarate reveal coordination of the ligand to the unique iron of the [4Fe-4S] cluster. (A) Wild type LmFH-2 in a complex with succinate (cyan). The distances from the T467 OH group to the succinate C3 (3.2 Å) and the water (coordinated to the [4Fe-4S] cluster) to the succinate C2 (2.9 Å) are shown as green dashed lines. (B) LmFH-2-R173A in a complex with fumarate (light orange). R173A is colored magenta. The distance from the T467 OH group to the fumarate C3 (3.3 Å) is shown as a green dashed line. (C) Superposition of LmFH-2 with succinate (cyan) and LmFH-2-R173A with fumarate (light orange). (D) The left panel shows a 2Fo – Fc electron density map contoured at 1.5 RMSD (blue mesh) for succinate (cyan), the [4Fe-4S] cluster, and a water molecule. The right panel shows a 2Fo – Fc electron density map contoured at 1.5 RMSD (blue mesh) for fumarate (light orange) and the [4Fe-4S] cluster. The [4Fe-4S] cluster is shown as orange (Fe) and yellow (S) spheres. The water molecule is shown as red spheres. (E) Dose–response curve of the inhibition of LmFH-2 by succinate against S-malate (left) and fumarate (right). Error bars represent three independent measurements.
Scheme 2
Scheme 2. Structures of Fumarate, S-Malate, Succinate, and Malonate
Figure 4
Figure 4
Malonate binds to the active site of LmFH-2 but not to the Fe–S cluster. (A) LmFH-2 in a complex with malonate (salmon). This structure contains an inactive [3Fe-4S] cluster. (B) Snapshot of the Fe–S cluster destruction by oxidation. LmFH-2 with malonate was exposed to molecular oxygen in the anaerobic chamber, which oxidizes the [4Fe-4S]2+ cluster to [3Fe-4S]+ and releases Fe (orange sphere; labeled in white). The 2Fo – Fc electron density map (blue mesh) is contoured at 1.5 RMSD for malonate (salmon), [3Fe-4S] cluster, and Fe. (C) EPR spectrum of oxygen-exposed LmFH-2 showing a signal characteristic of a [3Fe-4S]+ cluster with a g value of 2.02. (D) LmFH-2-T467A in a complex with malonate (salmon). This structure contains an active [4Fe-4S] cluster. The T467A substitution is colored light blue. (E) The 2Fo – Fc electron density map contoured at 1.5 RMSD (blue mesh) for malonate (salmon), [4Fe-4S] cluster, water molecule, and T467A substitution (light blue). (F) Superposition of LmFH-2-T467A in a complex with malonate (salmon) and LmFH-2 in a complex with succinate (cyan). The [3Fe-4S] cluster and [4Fe-4S] cluster are shown as orange (Fe) and yellow (S) spheres. The water molecules are shown as red spheres. The hydrogen bonds are shown as gray dashed lines.
Figure 5
Figure 5
Conformationally flexible LmFH-2 C-terminal domain. (A) Two views of the superposition of LmFH-2-holo dimers (chains A and B, C and D, E and F, and G and H) showing the C-terminal domain mobility. The N-terminal domain is colored light blue, and the C-terminal domain of each dimer is colored light pink (chains A and B), hot pink (chains C and D), purple (chains E and F), and violet (chains G and H). The [4Fe-4S] cluster is shown as orange (Fe) and yellow (S) spheres. (B) Another view of the LmFH-2-holo C-terminal domains showing its swiveling movement. (C) Table of RMSD values between LmFH-2 with the S-malate dimer and each LmFH-2-holo dimer. The Cα RMSD values were calculated using the ColorByRMSD PyMol script.
Figure 6
Figure 6
Proposed catalytic mechanism for class I FHs. In the dehydration of S-malate to fumarate, first, a proton is abstracted from S-malate C3 by T467 that is activated by either of the two arginine residues (R421 and R471), which accept the proton. In the second step, D135 donates a proton to the S-malate C2-hydroxyl group for elimination as water and subsequent formation of fumarate. The negative charge of D135 is stabilized by H334. In the hydration of fumarate to S-malate, D135 abstracts a proton from the water molecule bound to the [4Fe-4S] cluster to form a hydroxyl group for addition to C2 of fumarate, and then T467 and either of its partners R421 and R471 donate a proton to C3 of fumarate for subsequent formation of S-malate. The [4Fe-4S] cluster acts as a Lewis acid to activate the hydroxyl group from S-malate for elimination or water for addition.
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