PKU mutation G46S is associated with increased aggregation and degradation of the phenylalanine hydroxylase enzyme.

The G46S mutation in the phenylalanine hydroxylase (PAH) gene was identified by fluorescence-based single-strand conformation polymorphism (F-SSCP) analysis on phenylketonuria (PKU) haplotype 5.9 alleles. DNA sequencing of PAH exon 2 revealed a G-to-A transition in cDNA position 136. G46S mutations were present on 17 of 236 Norwegian PKU alleles (7.2%) and on 8 of 176 Swedish PKU alleles (4.5%). Analysis of all 13 exons with the flanking regions further detected a 1316-35c > t polymorphism (PAH intron 12), associated with both G46S and haplotype 5.9. Three patients were homozygous for the G46S mutation, two were untreated and had mild and severe mental retardation, respectively. The G46S mutation was introduced in the PAH cDNA by site-directed mutagenesis and expressed in three different systems (the pMAL/Escherichia coli system, the pcDNA3/human embryonic kidney (A293) cells, and the pcDNA3/TnT coupled in vitro transcription-translation system). The mutant recombinant E. coli fusion protein was recovered in high yield and with a specific activity of the purified tetrameric form, which was higher than the wild-type activity. After transient expression in A293 cells, the amount of the G46S protein was only about 3% of the wild type at equal PAH mRNA levels. The fusion protein cleaved by restriction protease factor Xa, as well as the enzyme produced by in vitro transcription-translation, revealed an abnormal susceptibility to form catalytically inactive high-molecular-mass aggregates of the enzyme. This aggregation, followed by an increased cellular degradation of the G46S mutant enzyme, is compatible with the clinical/metabolic phenotype of the affected homozygous and compound heterozygous patients.