The study and manipulation of the structures of mammalian phenylalanine hydroxylase (PAH)

Phenylalanine hydroxylase (PAH) is a liver enzyme critical for phenylalanine (Phe) homeostasis. Defective PAH results in aberrantly high Phe levels, the hallmark of phenylketonuria (PKU). Normally, Phe is both substrate and allosteric activator of PAH. Rising Phe promotes transient Phe binding to an ACT-ACT dimer, which stabilizes ACT domain dimerization, increases catalytic rate, and drives changes to multimerization state. We proposed that these rearrangements among the three domains of PAH support a Phe-sensitive dynamic equilibrium of architecturally distinct PAH conformations. We called these conformations resting-state ("RS-PAH") and activated ("A-PAH") PAH. RS-PAH dominates the equilibrium at low Phe, when PAH activity is low and ACT domains are not dimerized, while A-PAH dominates at high Phe, when PAH activity is highest and ACT domains are dimerized. Our goal has been to elucidate the structures of RS-PAH and A-PAH in order to understand normal PAH allostery and potentially improve function of PKU-associated PAH. Towards this goal, we 1) determined the first reported crystal structures of full length three-domain human and rat PAH, both in a RS-PAH conformation; 2) applied a suite of tools to monitor structural differences among PAH conformations; 3) took various approaches towards isolating the A-PAH conformation, including the design of several single-residue PAH variants. Two designed variants shift the conformational equilibrium towards A-PAH, making them improved candidates for the study of A-PAH using crystallography, SAXS and cryo-EM.