Structural and functional analysis of missense mutations in fumarylacetoacetate hydrolase, the gene deficient in hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease cause d by a deficiency of the enzyme involved in the last step of tyrosine degra dation, fumarylacetoacetate hydrolase (FAH), Thus far, 34 mutations in the FAH gene have been reported in various HT1 patients. Site-directed mutagene sis of the FAH cDNA was used to investigate the effects of eight missense m utations found in HTI patients on the structure and activity of FAH, Mutate d FAH proteins were expressed in Escherichia coli and in mammalian CV 1 cel ls. Mutations N16I, F62C, A134D, C193R, D233V, and W234G lead to enzymatica lly inactive FAH proteins. Two mutations (R341W, associated with the pseudo -deficiency phenotype, and Q279R) produced proteins with a level of activit y comparable to the wild-type enzyme. The N16I, F62C, C193R, and W234G vari ants were enriched in an insoluble cellular fraction, suggesting that these amino acid substitutions interfere with the proper folding of the enzyme. Based on the tertiary structure of FAH, on circular dichroism data, and on solubility measurements, we propose that the studied missense mutations cau se three types of structural effects on the enzyme: 1) gross structural per turbations, 2) limited conformational changes in the active site, and 3) co nformational modifications with no significant effect on enzymatic activity .