Research

Scientists from the IQFR, the Univ. of Bergen and CNIO report in PNAS the atomic structure of phenylalanine hydroxylase, a human enzyme responsible of the phenylketonuria disease.

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone.

An international team lead by Rocasolano Institute and University of Bergen (Norway) has been able to determine the atomic structure of PAH. The results of this work are published in the journal Proceedings of the National Academy of Sciences (PNAS).

In this work we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the pre-catalytic state. Crystal structures, solved at 3.18Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4. BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII. Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4. Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the pre-catalytic towards the active conformation, a molecular mechanism that was supported by site directed mutagenesis and targeted MD simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations

The present crystal structure of phenylalanine hydroxylase (PAH) provides the first and long-awaited 3D-structure of the full-length human PAH, both unbound and complexed with the tetrahydrobiopterin (BH4) cofactor.

Two decades after the first partial structures of human phenylalanine hydroxylase (PAH) were published, the results of a long-term and successful collaboration between researchers at Inst. Rocasolano (CSIC) and the University of Bergen in Norway and in collaboration with CNIO in Spain, can finally be presented: full-length structures of this very important metabolic enzyme. This work has been directed by Professors Juan A. Hermoso and Aurora Martinez with postdoctoral researchers Marte I. Flydal and Martin Alcorlo as shared first authors.

Reference:

Marte Innselset Flydal, Martin Alcorlo, Fredrik Gullaksen Johannessen, Siseth Martinez-Caballero, Lars Skjærven, Rafael Fernandez-Leiro, Aurora Martinez, Juan A. Hermoso. The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin. PNAS. DOI: 10.1073/pnas.1902639116.

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