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.
Highlights
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
The energy landscape obtained for WT-FAH and the mutant set identified two clearly distinct molecular mechanisms for pathogenicity: mutations located at the C-terminal domain (C-term) shift the equilibrium toward the monomer, triggering aggregation, whereas mutations located in the N-terminal domain (N-term) are likely to produce a significant conformational change that decreases the stability of monomeric FAH
The NMR measurement of the diffusion coefficient by diffusion ordered spectroscopy (DOSY; Fig. 1B) at 20 °C is consistent with a theoretical hydrodynamic diameter of 94 Å, in agreement with a dimer species for WT FAH
Summary
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. A reduced activity of FAH upon mutation leads to the accumulation of upstream metabolites like fumarylacetoacetate (FAA) and maleylacetoacetate, which are subsequently converted to succinylacetoacetate, decarboxylated to succinylacetone, and accumulated in many body tissues These metabolites are highly reactive with electrophilic compounds and responsible for the progressive hepatic, renal, and neurological damages [6]. We have thoroughly studied the monomer– dimer equilibrium of WT FAH by NMR spectroscopy and CD to show
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