Revisiting the Burden Borne by Fumarase: Enzymatic Hydration of an Olefin

Abstract

Fumarate hydratase (FH) is a remarkable catalyst that decreases the free energy of the catalyzed reaction by 30 kcal mol −1 , much larger than most exceptional enzymes with extraordinary catalytic rates. Two classes of FH are observed in nature: class-I and class-II, which have different folds, yet catalyze the same reversible hydration/dehydration reaction of the dicarboxylic acids fumarate/malate, with equal efficiencies. Using class-I FH from the hyperthermophilic archaeon Methanocaldococcus jannaschii (Mj) as a model along with comparative analysis with the only other available class-I FH structure from Leishmania major (Lm), we provide insights into the molecular mechanism of catalysis in this class of enzymes. The structure of MjFH apoprotein has been determined, revealing that large intersubunit rearrangements occur across apo-and holo-protein forms, with a largely preorganized active site for substrate binding. Site-directed mutagenesis of active site residues, kinetic analysis, and computational studies, including density functional theory (DFT) and natural population analysis, together show that residues interacting with the carboxylate group of the substrate play a pivotal role in catalysis. Our study establishes that an electrostatic network at the active site of class-I FH polarizes the substrate fumarate through interactions with its carboxylate groups, thereby permitting an easier addition of a water molecule across the olefinic bond. We propose a mechanism of catalysis in FH that occurs through transition-state stabilization involving the distortion of the electronic structure of the substrate olefinic bond mediated by the charge polarization of the bound substrate at the enzyme active site.

Enantioselectivity in the enzymatic dehydration of malate and tartrate: Mirror image specificities of structurally similar dehydratases

“…Apart from activity on (S)-malate (Bellur et al, 2023), as shown in Figure S2A, MjFH was found to be active on (S,S)-tartrate corroborating previous reports on other Class-I FHs . The enzyme was inactive on (R,R)-tartrate, and (R)-malate.…”

“…This apart G216 of the KGXGS motif contacts C2  OH (Figure 4a). All these residues superpose well on the structure of Mj FH (Bellur et al, 2023). Examination of the active site in the modeled structure of Ec L‐TTD did not reveal any difference in the orientation of the ligand‐binding and catalytic residues, that could account for the altered substrate stereospecificity preference in L‐TTD compared with Class‐I FHs (Figure 4a,b).…”

“…Generation of expression plasmids carrying genes for Mj FH α and β subunits has been described (Bellur et al, 2023). Expression and purification of Mj FH were carried out as previously described (Bellur et al, 2023).…”

“…Generation of expression plasmids carrying genes for MjFH α and β subunits has been described (Bellur et al, 2023). Expression and purification of MjFH were carried out as previously described (Bellur et al, 2023). Genes for the two subunits, L-TTDα and L-TTDβ were PCR amplified using primers listed in Table S1 from the genomic DNA of the E. coli DH5α strain and cloned into two tandem multiple cloning sites of the pET-Duet vector using the enzymes EcoRI and HindIII for L-TTDα such that the (His) 6 tag is at its N-terminus and enzymes NdeI and XhoI for L-TTDβ.…”

“…Although preliminary characterization of L‐TTD from Pseudomonas putida (Kelly & Scopes, 1986), Escherichia coli (Reaney et al, 1993) and an L‐tartrate fermenting anaerobic bacteria (Schink, 1984) has been reported, no structure of L‐TTD is available. Class‐I FHs have been investigated extensively with crystal structures of the enzyme from Leishmania major ( Lm ) (Feliciano et al, 2016) and Methanocaldococcus jannaschii ( Mj ) (Bellur et al, 2023) showing identical active site residues in similar conformation, the roles of which in substrate binding and catalysis have been confirmed by extensive mutagenesis. Due to the high sequence similarity, the modeled structure of L‐TTD is similar to FH with identical active site architecture.…”