The structure of full-length human phenylalanine hydroxylase in complex with tetrahydrobiopterin

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 (BH4) that partly acts as a pharmacological chaperone. Here 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.Significance StatementThe 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.The BH4-bound state is physiologically relevant, keeping PAH stable and in a pre-catalytic state at low L-Phe concentration. Furthermore, a synthetic form of BH4 (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 BH4, which aid to understand the stabilization by BH4 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-4605This article contains supporting information.

The Structure of Human Tyrosine Hydroxylase Reveals the Mechanism for Feedback Inhibition by Dopamine

“…Similar to what was found in the recent cryoEM PAH structure 10 , there was nonisotropy in the resolution, with the RDs showing less resolution both because of their inherent flexibility and the dynamic linkers that connect them to the central structure.…”

“…By comparing the maps for TH ( Figs. 1 and 2) and PAH 10 Supplementary Fig. 10d, right), and shifted 88° compared to the TH monomer ( Supplementary Fig.…”

“…1a): an N-terminal regulatory domain (RD) that consists of a structured ACT (aspartate kinasechorismate mutase-TyrA) domain preceded by a less structured N-terminal tail of varying length; a central catalytic domain (CD) that contains the active site iron and binding-sites for substrate and cofactor; and a C-terminal oligomerization domain (OD) responsible for dimerization and tetramerization 6,7 . The full-length structures of rat and human PAH have recently been solved by X-ray crystallography [8][9][10] and cryoEM 10 . For human and rat TH and human TPH, crystal structures encompassing CD+OD are available 11,12 .…”

Second-Generation Pharmacological Chaperones: Beyond Inhibitors

“…Activation of PAH by l -Phe occurs at high concentrations of l -Phe in blood while negative regulation by BH4 takes place at a low level. Co-crystallizations of PAH with BH4 or l -Phe allowed the fine understanding of these regulation mechanisms [ 97 , 98 , 99 , 100 ]. PAH protein exists as a mixture of native structures in “resting” or in “activated” states depending on the l -Phe concentration levels [ 97 ].…”

“…The PC behaviour of BH4 was highlighted by Erlandsen et al, on fifteen BH4-responsive mutants [ 107 ]. The recent co-crystallization of the full-length h PAH with BH4 was reported, helping to understand the stabilization by the cofactor (PDB code 6HYC) [ 98 ]. Authors showed that the stabilization of the protein by BH4 is initiated at its binding-site in the catalytic domain but also affects the whole structure of the protein.…”