Structural basis for ligand-dependent dimerization of phenylalanine hydroxylase regulatory domain

Abstract

The multi-domain enzyme phenylalanine hydroxylase (PAH) catalyzes the hydroxylation of dietary I-phenylalanine (Phe) to I-tyrosine. Inherited mutations that result in PAH enzyme deficiency are the genetic cause of the autosomal recessive disorder phenylketonuria. Phe is the substrate for the PAH active site, but also an allosteric ligand that increases enzyme activity. Phe has been proposed to bind, in addition to the catalytic domain, a site at the PAH N-terminal regulatory domain (PAH-RD), to activate the enzyme via an unclear mechanism. Here we report the crystal structure of human PAH-RD bound with Phe at 1.8 Å resolution, revealing a homodimer of ACT folds with Phe bound at the dimer interface. This work delivers the structural evidence to support previous solution studies that a binding site exists in the RD for Phe, and that Phe binding results in dimerization of PAH-RD. Consistent with our structural observation, a disease-associated PAH mutant impaired in Phe binding disrupts the monomer:dimer equilibrium of PAH-RD. Our data therefore support an emerging model of PAH allosteric regulation, whereby Phe binds to PAH-RD and mediates the dimerization of regulatory modules that would bring about conformational changes to activate the enzyme.

The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer

“…Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18]. This model is supported by recent studies of the isolated regulatory domain[14, 19, 20] and of the intact protein[13, 18]. TyrH is activated by phosphorylation of Ser40 in its N-terminal regulatory domain and inhibited by catecholamines[10, 21].…”

“…The resting form of PheH is inactive, with the N-terminus of the regulatory domain partially occluding the active site[12]. Phenylalanine binding to an allosteric site in the regulatory domain[13, 14] causes a significant conformational change that opens up the active site[15, 16]. Key to this conformational change is formation of an ACT domain dimer by regulatory domains from two subunits[17, 18].…”

Phenylketonuria felnőttkorban

“…A PAH gén közel 1000 mutációját írták eddig le, ezeknek 60%-a egyetlen nukleotidot érintő pontmutáció [10]. A PAH-mutációk, valamint klinikai jelentőségük a BIOPKUdb online adatbázisán keresztül elérhetők (http://www.biopku.org).…”

“…Sok mutációval kapcsolatban már ismert a genotípus-fenotípus korreláció, azaz milyen a residualis enzimaktivitás vagy az illető fogékonysága különböző terápiás lehetőségekre. Magyarországon a leggyakoribb genetikai eltérés a körülbelül 75%-ban elő-forduló R408W-pontmutáció, amely klasszikus PKU-t eredményez közel 0%-os enzimaktivitással [4,10,11].…”

Substituting Tyr138 in the active site loop of human phenylalanine hydroxylase affects catalysis and substrate activation

“…The first model was originally based on indirect enzyme kinetic 4 and biophysical studies on the rPAH homotetramer and truncated RD constructs, but has lately gained further support from the determination of th e high resolution crystal structure (PDB ID: 5FII at 1.8 Å) of a homodimeric truncated form of the human RD (hPAH‐RD) 16. Representing the key finding of this study, the structure revealed two l ‐Phe molecules bound to a homodimer at the interface of the two β 1 α 1 β 2 β 3 α 2 β 4 ACT domain folds along the plane of the twofold axis 16. Although the crystal structure of an enzyme·substrate complex in the full‐length homotetramer is still not available, our multiple crystal structures [see table in Ref.…”