Structural, biochemical and biophysical characterization of recombinant human fumarate hydratase

Abstract

Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l‐malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X‐ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221, with unit‐cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C‐terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES‐binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

Fumarase From Cyanidioschyzon merolae Stably Shows High Catalytic Activity for Fumarate Hydration Under High Temperature Conditions

“…It is suggested that TeFum also becomes active at these growth temperatures. The optimum pH for CmFUM (pH 7.5 for fumarate hydration; pH 8.5 for l-malate dehydration) and TeFum (pH 7.0 for fumarate hydration; pH 7.5 for l-malate dehydration) were approximately the same as those of Class ІІ Fums from other organisms (pH 6.5-8.5, seven species: R. oryzae, Synechocystis 6803, Saccharomyces cerevisiae, S. solfataricus, C. glutamicum, A. thaliana, and Homo sapience; Puchegger et al, 1990;Keruchenko et al, 1992;Genda et al, 2006;Song et al, 2011;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019; Figures 2B,D). Intercellular pH of C. merolae and cyanobacteria is maintained near neutral where CmFUM and TeFum show enzymatic activities (Coleman and Colman, 1981;Zenvirth et al, 1985;Mangan et al, 2016).…”

“…The K m of CmFUM (0.27 mM) and TeFum (0.14 mM) for fumarate were within the range of most Class ІІ enzymes (0.03-3.07 mM, nine species: E. coli, C. glutamicum, R. oryzae, Synechocystis 6803, S. cerevisiae, S. solfataricus, A. thaliana, T. thermophilus, and H. sapience;Woods et al, 1988;Puchegger et al, 1990;Keruchenko et al, 1992;Mizobata et al, 1998;Song et al, 2011;Lin et al, 2018;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019; A B…”

“…Katayama et al, 2019; Table 1). The k cat of CmFUM (235 s −1 ) and TeFum (37 s −1 ) for fumarate were within the range of Class ІІ Fums (21-513 s −1 , four species: C. glutamicum, Synechocystis 6803, A. thaliana, and H. sapience, Lin et al, 2018;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019; Table 1). The k cat /K m of CmFUM (872 s −1 mM −1 ) for fumarate was similar to that of Class ІІ Fum from H. sapience (850 s −1 mM −1 ; Ajalla Aleixo et al, 2019), and higher than those of cyanobacterial Class ІІ Fums (Synechocystis 6803: 415 s −1 mM −1 , TeFum: 278 s −1 mM −1 ; Katayama et al, 2019) and Class ІІ Fums from C. glutamicum (247 s −1 mM −1 ; Lin et al, 2018) and A. thaliana (30 s −1 mM −1 ; Zubimendi et al, 2018; Table 1).…”

“…Like Class ІІ Fums from other organisms (A. thaliana, Synechocystis 6803, C. glutamicum, and H. sapience; Genda et al, 2006;Zubimendi et al, 2018;Ajalla Aleixo et al, 2019;Katayama et al, 2019), CmFUM and TeFum preferentially catalyze fumarate hydration rather than l-malate dehydration ( Table 1). The K m of CmFUM (0.27 mM) and TeFum (0.14 mM) for fumarate were within the range of most Class ІІ enzymes (0.03-3.07 mM, nine species: E. coli, C.…”

The Landscape of Mutations in Human Fumarate Hydratase

“…We do not consider Site B due to the unknown and conflicting evidence surrounding its importance. For this study we chose to focus on the crystal structure 5upp 24 , which covers residues 49-510 of the 510 residue protein assembled into a homotetramer.…”