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.The human phenylalanine hydroxylase (hPAH) catalyzes the hydroxylation of l-phenylalanine (l-Phe) into l-tyrosine (l-Tyr). The reaction is the first step in the catabolic pathway of l-Phe/l-Tyr and proceeds to feed neurotransmitter biosynthetic pathways. In non-pathological conditions, degradation of excessive l-Phe by hPAH sustains physiological plasmatic levels of l-Phe (<120 µM) 1 . Deficiency in hPAH leads to phenylketonuria (PKU), characterised by a toxic accumulation of l-Phe and depletion of precursors for neurotransmitter biosynthesis in the central nervous system that overall result in cognitive disability and neurological impairment. Despite being the most prevalent disorder of the amino acid metabolism, the pathophysiology of PKU remains to be fully elucidated and treatment options are mostly limited to a life-long l-Phe-restricted diet 1 . PKU is caused by mutations in the PAH gene, most being missense mutations that affect folding, catalysis and/or regulation of the enzyme 2,3 . Understanding the structural and regulatory properties of hPAH is essential to outline pathophysiological mechanisms in PKU and delineate novel therapeutic strategies. However, the difficult manipulation of recombinant hPAH has thus far hindered its structural characterisation. hPAH is a member of the aromatic

Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One Derivatives

“…Incubation with 100 μM L-Phe in the presence of 1% DMSO decreased the proteolytic rate (kP) from 0.41 ± 0.03 to 0.15 ± 0.01 min −1 (Figure 6A,F) in line with our previous observation [8]. With respect to the control sample, compounds 3 and 8 exerted a protective effect as the kP decreased to 0.29 ± 0.04 and 0.30 ± 0.01 min −1 (Figure 6B,C,F), respectively, Incubation with 100 µM L-Phe in the presence of 1% DMSO decreased the proteolytic rate (k P ) from 0.41 ± 0.03 to 0.15 ± 0.01 min −1 (Figure 6A,F) in line with our previous observation [8].…”

“…The effect of compounds 3, 8, 9, and 11 on the hPAH steady-state kinetics was studied over a substrate range of 25 µM to 2.5 mM L-Phe. The obtained hPAH kinetic parameters (V max , S 0.5 , h and catalytic efficiency) for the control sample in the presence of 1% DMSO were in the range of those described in the literature [8,34] (Table 1, Reaction buffer), including the reported substrate inhibition ( Figure 4A). In the presence of compounds 3, 8, 9, and 11, inhibition of enzyme activity at high L-Phe concentrations was maintained for compounds 8, 9, and 11 ( Figure 4A).…”

Manipulation of a Cation-π Sandwich Reveals Conformational Flexibility in Phenylalanine Hydroxylase

“…One such SAXS-supported A-PAH conformation model is illustrated ( Fig 1C) (13); it places the dimerized regulatory domain considerably farther from the protein's center of mass relative to our original, other previous, and contemporary models (5,7,11,24).…”

“…PDB: 5DEN, 5FGJ, 6N1K) (5,11,13). Alternate models exist for the A-PAH conformation, some of which are supported by small angle X-ray scattering (SAXS) data, and a crystal structure of allosteric Phe bound to a portion of the protein (residues 34-111, segment 3, the ACT domain, PDB: 5FII) (4,5,7,11,13,24).…”

“…PDB: 5DEN, 5FGJ, 6N1K) (5,11,13). Alternate models exist for the A-PAH conformation, some of which are supported by small angle X-ray scattering (SAXS) data, and a crystal structure of allosteric Phe bound to a portion of the protein (residues 34-111, segment 3, the ACT domain, PDB: 5FII) (4,5,7,11,13,24). One such SAXS-supported A-PAH conformation model is illustrated (Fig 1C) (13); it places the dimerized regulatory domain considerably farther from the protein’s center of mass relative to our original, other previous, and contemporary models (5,7,11,24).…”