ORCID Profile
0000-0002-1862-8950
Current Organisation
Spanish National Research Council
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Publisher: Cold Spring Harbor Laboratory
Date: 15-02-2019
DOI: 10.1101/552281
Abstract: 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 (BH 4 ) that partly acts as a pharmacological chaperone. Here we present the first structures of full-length human PAH (hPAH) both unbound and complexed with BH 4 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 BH 4 . BH 4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe II . Upon BH 4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH 4 . Importantly, BH 4 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 BH 4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH 4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH 4 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 (BH 4 ) cofactor. The BH 4 -bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH 4 (Kuvan®) is the only drug-based therapy for a subset of phenylketonuria patients. We found two tetramer conformations in the same crystal, depending on the active site occupation by BH 4 , which aid to understand the stabilization by BH 4 and the allosteric mechanisms in PAH. The structure also reveals the increased mobility of human-compared with rat PAH, in line with an increased predisposition to disease-associated mutations in human. Classification : Biological Science (major), biochemistry (minor) The authors declare no conflict of interest. Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.wwpdb.org (PDB ID codes 6HYC and 6HPO). The EM maps have been deposited in the EMDB with accession s EMD-4605 This article contains supporting information.
Publisher: Proceedings of the National Academy of Sciences
Date: 22-05-2019
Abstract: 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 (BH 4 ) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH 4 in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH 4 . BH 4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic Fe II . Upon BH 4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH 4 . Importantly, BH 4 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 BH 4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH 4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH 4 from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics 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.
No related grants have been discovered for Juan Hermoso.