Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability.

ABSTRACT

Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.