Hereditary tyrosinemia type I-associated mutations in fumarylacetoacetate hydrolase reduce the enzyme stability and increase its aggregation rate

Abstract

More than 100 mutations in the gene encoding fumarylacetoacetate hydrolase (FAH) cause hereditary tyrosinemia type I (HT1), a metabolic disorder characterized by elevated blood levels of tyrosine. Some of these mutations are known to decrease FAH catalytic activity, but the mechanisms of FAH mutation-induced pathogenicity remain poorly understood. Here, using diffusion ordered NMR spectroscopy, cryo-EM, and CD analyses, along with site-directed mutagenesis, enzymatic assays, and molecular dynamics simulations, we investigated the putative role of thermodynamic and kinetic stability in WT FAH and a representative set of 19 missense mutations identified in individuals with HT1. We found that at physiological temperatures and concentrations, WT FAH is in equilibrium between a catalytically active dimer and a monomeric species, with the latter being inactive and prone to oligomerization and aggregation. We also found that the majority of the deleterious mutations reduce the kinetic stability of the enzyme and always accelerate the FAH aggregation pathway. Depending mainly on the position of the amino acid in the structure, pathogenic mutations either reduced the dimer population or decreased the energy barrier that separates the monomer from the aggregate. The mechanistic insights reported here pave the way for the development of pharmacological chaperones that target FAH to tackle the severe disease HT1.

Keywords: biophysics; enzyme mutation; fumaryl acetoacetate hydrolase; nuclear magnetic resonance (NMR); protein aggregation; protein stability; rare disease; tyrosine; tyrosinemia type I.

Figure 1.

A, substrate conversion as a function of the total concentration of FAH (C_T). The black dashed line represents the reported K_D value that is consistent with the data. Red and green dashed lines fit the subset of data in which the majoritarian species correspond to the monomer or the dimer, respectively. B, overlap of DOSY spectra for WT FAH at different temperatures, as indicated. Spectra have been normalized to Tris and 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) because both molecules show no structural variation in this temperature range. The lines reflect the diffusion coefficient (log D, y axis), whereas the circles indicate the positions of the peaks.

Figure 2.

A and D, CD data for WT FAH at concentrations where the monomer (A) or the dimer (D) species are majoritarian. Purple open circles correspond to the experimental data, whereas solid black lines are the best collective fitting to the bimodal thermal denaturation. B, representative micrograph from aggregates of WT FAH. The inset reflects the average length of the aggregating particles from the green-highlighted stretch of particles. C, Arrhenius plot for WT FAH aggregation constant (k_Ag) versus temperature. The solid line corresponds to the best fit of the magnitudes shown in the axes. E, plot of the monomer concentration versus the total concentration (C_T) of WT FAH. The lines where the FAH concentration is monomeric (M) or aggregated (Ag) are shown in green.

Scheme 1

Figure 3.

Biochemical and biophysical properties of the investigated mutants. A, enzyme activity (E.A.). B, expression (Exp.) yield. C, unfolding free energy. D, dimer content. E, aggregation rate of the monomeric species. All the properties are relative values from WT FAH unless otherwise indicated. WT/mutant values are indicated from black/green bars, respectively. The error bars were obtained from duplicate data and error propagation. F, fraction of dimer (as compared with WT FAH) plotted in the dimer structure of FAH. The color code is indicated in the legend. G, plot of the monomer concentration versus the total concentration (C_T) of representative mutants: F62C (black squares), WT (purple circles), N16I (green diamonds), G158D (crosses), W234G (purple triangles), V166G (black stars), and G337S (green triangles). The asterisk indicates that the data are not available.

Figure 4.

A–C, human fibroblastoid M1 cells 24 h after FAH-GFP transfection as measured by FACS. For each FAH variant, the left bar plot shows the percentage of fluorescent cells from two independent experiments, whereas the histograms show one representative data set. FITC-A shows GFP fluorescence emission; the percentage and median fluorescence intensity of GFP-positive cells are indicated in each condition. Fluorescence microscopy images show representative examples of the protein distribution in the cellular environment. The images show the overlap of the 4′,6′-diamino-2-phenylindole channel (blue) with the GFP-sensitive excitation frequency (green). Transfection with WT FAH-GFP construct (A), Gr1 group mutants (V166G and W234G, B), and Gr2 group mutants (A35T and T294P, C) is shown. D, increase in fluorescence for the M1 FAH-GFP clones after treatment with MG-132. The increase in the fluorescence is due to protein accumulation in the cell caused by proteasome degradation pathway inhibition and is related to the WT FAH basal fluorescence in the absence of proteasomal inhibitor.

Figure 5.

Energy diagram for WT and M1V FAH (solid line), the mutants of the Gr1 group (purple dashed line) and the mutants of the Gr2 group (dashed green line). D, dimer; M, monomer (folded); Ag, aggregate. The mutant G207D destabilizes the monomeric form, as indicated. The free energy for the transition state toward aggregation has been calculated from the experimental energy barrier by means of the Eyring equation, whereas the other changes in energy levels are only qualitative estimations, are mutant-dependent, and are not drawn to scale.