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

Catarina S. Tomé,Pedro M. F. Sousa,Paula Leandro,Haydyn D. T. Mertens,João B. Vicente,João Leandro,Mariana P. Amaro,Raquel R. Lopes

doi:10.1038/s41598-019-49944-x

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.

Highlights

Human phenylalanine hydroxylase hydroxylates l-phenylalanine (l-Phe) to l-tyrosine, a precursor for neurotransmitter biosynthesis
N-terminally His6-tagged full-length tetrameric Human phenylalanine hydroxylase (hPAH) were analyzed by activity assays. hPAH activity was determined as a function of l-Phe concentration (Fig. 1A) and the data were fitted with a modified Hill equation accounting for substrate inhibition[31], allowing to estimate the Vmax (4688 ± 120 nmol l-Tyr·min−1·mg−1), S0.5 (107 ± 6 μM), h (1.8 ± 0.1) and catalytic efficiency (Kcat/S0.5 = 3.01 μM−1·min−1)
The availability of structural data for full-length human phenylalanine hydroxylase has far been hampered by the inability to produce a conformationally homogeneous tetrameric preparation

Summary

Introduction

Human phenylalanine hydroxylase (hPAH) hydroxylates l-phenylalanine (l-Phe) to l-tyrosine, a precursor for neurotransmitter biosynthesis. Solution structural analyses of rat PAH confirmed distinct conformations for the inactive and l-Phe-activated enzymes and supported dimerisation of regulatory domains as the substrate activation mechanism[13,15]. Our low resolution solution structures determined by small-angle X-ray scattering disclose a conformational transition from the inactive state (that agrees with crystallographic observations) to an active state where regulatory domains associate above the four-helix bundle. These structures, combined with biophysical data obtained for resting and activated states, validate the model of l-Phe allosteric activation and elucidate the regulatory mechanism of hPAH

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Results

Conclusion