tertiary structure</scene> of each monomer of phenylalanine hydroxylase is organized from 2 alpha helices and 4 beta-strands into an alpha-beta sandwich motif (BaBBaB fold). The structural motif of an alpha-beta sandwich motif has the 4 antiparallel beta-strands flanked on one side by the 2 alpha-helices (3 & 5). The tertiary structure of a phenylalanine hydroxylase protein is built from an N-terminal regulatory domain (residues 1-117), a catalytic domain (residues 118-410), and a tetramerization domain (residues 411-452) (3 & 6). The catalytic domain includes the binding sites for iron, substrate, and cofactor. The binding sites are at residues 285, 290, and 330. The archetypical (ACT) domain is in the N-terminal regulatory domain where the proposed enzyme binding to an allosteric site (residues 3-11) (6).

Quaternary Structure:

The quaternary structure of PAH is a , a of dimers. It is a multidomain, homo-oligomeric protein with dihedral (D2) symmetry (3 & 6).

Substrate & Catalysis

The substrate of phenylalanine hydroxylase is the amino acid L-phenylalanine. Phenylalanine binds between the regulatory domain and the interacting , near the sequence binding motif. The activation of phenylalanine hydroxylase by L-phenylalanine induces a large conformational change, but a slow global conformational change. Full activation of PAH involves the shift and dimerization of the regulatory domains (6). PAH is an iron (Fe2+) containing enzyme. The iron binds to 2 histidines at the active site (3 & 6). The of PAH include 6R-L-erythro-tetrahydrobiopterin (BH4) and oxygen (3). is sandwiched between hydrophobic residues and forms several hydrogen bonds with the N-terminal autoregulatory tail. BH4 binding causes a limited conformational change (mostly constrained to the N-terminal tail). PAH lacking this tail is not regulated by either BH4 or L-phenylalanine and is constitutively active. The BH4 binding site is flanked by the N-terminal (residues 21-32), the active-site lid (130-150), the Fe+2-coordinating residues, the Beta 6-alpha 7 loop (residues 245-251), and F254 (6). Tetrahydrobiopterin induces a negative heterotropic allosteric effect on the enzyme, which is observed as the activation rate is slower for the BH4 holoprotein than compared to the unbound enzyme. Prior to BH4 binding, (PAH unbound state) a polar and salt-bridge interaction network links the three PAH domains.

Function

The genetic information that codes for the production of phenylalanine hydroxylase is found on the long arm of chromosome 12 and contains 13 exons (1, 7, & 8). PAH is a metabolic enzyme contained in liver cells that catalyzes the hydroxylation reaction of the amino acid L-Phenylalanine to L-Tyrosine (2, 4, & 9). This protein specifically catalyzes the rate-limiting step in the phenylalanine catabolism, which is the para-hydroxylation step of the aromatic side chain (4 & 9). This catalysis is done by hydroxylation of its substrate by incorporation of one oxygen atom into the aromatic ring, and the final reaction includes the reduction of the second oxygen atom to water using electrons supplied by tetrahydrobiopterin (BH4). BH4 functions as a co-substrate that is hydroxylated at each turnover to pterin-4a-carbinolamine (4a-OH-BH4), with consequent dissociation from the enzyme (4). The major regulatory mechanisms of phenylalanine hydroxylase include activation of phenylalanine inhibition by BH4 and additional activation by (5). Phosphorylation acts as a mediator of phenylalanine activation by decreasing the phenylalanine concentration required to activate enzyme phosphorylation at Ser16 (4). For reference, an image of the enzyme was also included. Substrate activation and positive homotropic allosteric for phenylalanine binding involve all three functional domains and all four subunits in the holoenzyme (5). The hypothesized cause of the phenylalanine activation mechanism is that the homotropic binding of phenylalanine at the active site and the regulatory domain is involved in cooperativity through the interactions with the catalytic and oligomerization domains. Phenylalanine binds to an allosteric site, beside the active site, on the regulatory domain, which induces large conformational changes (1 & 6). Allosteric regulation is necessary to maintain phenylalanine levels below neurotoxic levels (4). BH4 acts as a negative allosteric regulator by blocking phenylalanine activation, however, BH4 binding to a Phe-activated form of PAH results in positive cooperativity.

Phenylketonuria

L-Tyrosine is the precursor to neurotransmitters such as epinephrine, dopamine, and serotonin (8). It is essential that L-phenylalanine is converted into L-tyrosine by the hydroxylation reaction. In order for this conversion to be successful, the enzyme phenylalanine hydroxylase needs to be able to function properly. PAH depletion or leads to excessive accumulation of toxic L-Phe levels. However, normal physiological plasmatic levels of L-phenylalanine are less than 120 micromolar (4). Not only does dysfunctional PAH lead to increased phenylalanine levels in the blood, but it also has the appearance of urine metabolites that arise from the transamination of L-Phe to phenylpyruvate (4). When the enzyme PAH doesn’t function correctly, the autosomal recessive metabolic disorder Phenylketonuria (PKU) occurs. PKU is a congenital disorder characterized by excessive amounts of L-phenylalanine that buildup to neurotoxic amounts leading to cognitive disability and neurological impairment, including profound mental retardation, seizures, microcephaly, and delayed development (2, 3, & 6). The severity of PKU is dependent upon the severity of the enzyme’s mutation (3 & 10). PAH mutations result in reduced enzyme activity and stability and some alter their oligomeric state (6). These mutations spread throughout the 3D structure, but most are located in the catalytic domain (10). Loss of enzymatic function is caused mainly by folding defects that lead to decreased protein stability (6). The mutation leads to a truncated form of the final 52 amino acids. These C-terminal amino acids are a part of the tetramerization domain. Another frequent mutation is a CGG-to-TGG transition on exon 12. This mutation leads to a substitution of Arginine for Tryptophan at position 408. This missense mutation results in undetectable levels of phenylalanine and the severe PKU phenotype (10). Treatments for phenylketonuria include a lifelong diet avoiding foods containing phenylalanine and supplementation of synthetic formations of the cofactor tetrahydrobiopterin (BH4) (11). Testing for PKU can be done early on in the lifespan to determine if the disease is present and to start avoiding foods containing phenylalanine (3).

References

1. Gjetting, T.; Petersen, M.; Guldberg, P.; Güttler, F. In Vitro Expression of 34 Naturally Occurring Mutant Variants of Phenylalanine Hydroxylase: Correlation with Metabolic Phenotypes and Susceptibility toward Protein Aggregation. Mol. Genet. Metab 2001, 72 (2), 132–143. https://doi.org/10.1006/mgme.2000.3118.

2. Waters, P. J. HowPAH Gene Mutations Cause Hyper-Phenylalaninemia and Why Mechanism Matters: Insights from in Vitro Expression. Hum. Mutat 2003, 21 (4), 357–369. https://doi.org/10.1002/humu.10197.

3. Siltberg-Liberles, J.; Steen, I. H.; Svebak, R. M.; Martinez, A. The Phylogeny of the Aromatic Amino Acid Hydroxylases Revisited by Characterizing Phenylalanine Hydroxylase from Dictyostelium Discoideum. Gene 2008, 427 (1-2), 86–92. https://doi.org/10.1016/j.gene.2008.09.005.

4. Flydal, M. I.; Martinez, A. Phenylalanine Hydroxylase: Function, Structure, and Regulation. IUBMB Life 2013, 65 (4), 341–349. https://doi.org/10.1002/iub.1150.

5. Carluccio, C.; Fraternali, F.; Salvatore, F.; Fornili, A.; Zagari, A. Structural Features of the Regulatory ACT Domain of Phenylalanine Hydroxylase. PLoS ONE 2013, 8 (11), e79482. https://doi.org/10.1371/journal.pone.0079482.

6. Flydal, M. I.; Alcorlo-Pagés, M.; Johannessen, F. G.; Martínez-Caballero, S.; Skjærven, L.; Fernandez-Leiro, R.; Martinez, A.; Hermoso, J. A. Structure of Full-Length Human Phenylalanine Hydroxylase in Complex with Tetrahydrobiopterin. Proc. Natl. Acad. Sci. U.S.A. 2019, 116 (23), 11229–11234. https://doi.org/10.1073/pnas.1902639116.

7. Scriver, C. R. ThePAH Gene, Phenylketonuria, and a Paradigm Shift. Hum. Mutat 2007, 28 (9), 831–845. https://doi.org/10.1002/humu.20526.

8. Shebl, G.; Ahmed, H.; Kato, A.; Dawoud, H.; Hamza, M.; Haider, A. Detection of Sequence Mutations in Phenylalanine Hydroxylase (PAH) Gene Isolated from Egyptian Phenylketonuria (PKU) Patients. Egypt. J. Exp. Biol. (Bot.) 2019, 15 (2), 295. https://doi.org/10.5455/egyjebb.20190804010102.

9. Matthews, D. E. An Overview of Phenylalanine and Tyrosine Kinetics in Humans. J Nutr. 2007 Jun; 137(6 Suppl 1): 1549S–1575S. doi: 10.1093/jn/137.6.1549S

10. Fusetti, F.; Erlandsen, H.; Flatmark, T.; Stevens, R. C. Structure of Tetrameric Human Phenylalanine Hydroxylase and Its Implications for Phenylketonuria. J. Biol. Chem 1998, 273 (27), 16962–16967. https://doi.org/10.1074/jbc.273.27.16962.

11. Blau, N.; Erlandsen, H. The Metabolic and Molecular Bases of Tetrahydrobiopterin-Responsive Phenylalanine Hydroxylase Deficiency. Mol. Genet. Metab 2004, 82 (2), 101–111. https://doi.org/10.1016/j.ymgme.2004.03.006.