Characterization of phenylalanine hydroxylase from Caenorhabditis elegans. Insights into the evolution of human activity and regulation

Abstract

Phenylalanine hydroxylase (PAH) is an iron dependent enzyme that catalyzes the L-Phe to L-Tyr hydroxylation using tetrahydrobiopterin (BH4) as natural cofactor and O2 as cosubstrate. Mutations in the human PAH (hPAH) gene are associated to phenylketonuria. hPAH activity is tightly regulated by different mechanisms, including activation by L-Phe and phosphorylation, and inhibition by BH4. These regulatory effects are mediated by ligand induced changes in intra- and inter-subunit interactions in the tetramer. PAH enzymes from lower organisms have also been studied in some detail, but their catalytic and regulatory properties are unknown. We report the expression, purification and molecular characterization of a tetrameric PAH from C. elegans (cePAH). Our data show that cePAH and hPAH display similar specific activity, apparent affinities for L-Phe, BH4 and O2, secondary structure and thermal stability, but substantially diverges in its regulatory properties. Thus, cePAH did not require incubation with L-Phe to display maximal activity and also lacked positive cooperativity for the substrate. These results appear to be related to structural differences between both enzymes. Our results also provide further insights into the physiological role of PAH in lower organisms and the changes in PAH architecture, activity and regulation along evolution. This research was supported by the Research Council of Norway