Missense mutations in the N-terminal domain of human phenylalanine hydroxylase interfere with binding of regulatory phenylalanine

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH ) is an autosomal recessive disorder caused by >400 mutations in the PAH ge ne. Recent work has suggested that the majority of PAH missense mutations i mpair enzyme activity by causing increased protein instability and aggregat ion. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory do main of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacte rial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residu es 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To ex amine whether N-terminal PAH mutations affect the ability of PAH to bind ph enylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V , T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2-120 ) of human PAH were expressed as fusion proteins in Escherichia coli. Bindi ng studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced th is phenylalanine-binding capacity. Our data suggest that impairment of phen ylalanine-mediated activation of PAH may be an important disease-causing me chanism of some N-terminal PAH mutations, which may explain some well-docum ented genotype-phenotype discrepancies in PAH deficiency.