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

Torben Gjetting, Marie Petersen, Per Guldberg, Flemming Güttler

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. 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 domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (GAL) and 65-69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine 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. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency.
Keyword: ESCHERICHIA-COLI,MUTASE-PREPHENATE DEHYDRATASE,PROTEIN,MECHANISM,PHENYLKETONURIA,GENE PAH,GENOTYPE-PHENOTYPE CORRELATIONS,CRYSTAL-STRUCTURE,FEEDBACK INHIBITION,STRUCTURAL BASIS
Original languageEnglish
JournalAmerican Journal of Human Genetics
Volume68
Issue number6
Pages (from-to)1353-1360
ISSN0002-9297
DOIs
Publication statusPublished - 2001
Externally publishedYes

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