Catalytic Domain of Phenylalanine Hydroxylase (PDB
ID: 6PAH) from Homo sapiens
Created by: Jennifer Ahn
Phenylalanine hydroxylase (PDB ID: 6PAH) is an important biological catalyst for breaking down L-phenylalanine (L-phe) in Homo sapiens. Excess L-phe is converted to L-tyrosine (L-tyr) by para-hydroxylation of the aromatic side chain. Along with natural cofactors 6(R)-l-erythro-tetrahydrobiopterin (BH4) and dioxygen (O2), phenylalanine hydroxylase (PAH) initiates the rate-determining step of L-phe catabolism (1-3). L-phe is an essential amino acid, which must be consumed in one’s diet. Dysfunctional or deficient amounts of PAH can result in accumulated L-phe in the bloodstream. This leads to a series of neurological complications, including mental retardation, seizures, and phenylketonuria (PKU) (1).
PKU is a recessive genetic disorder caused by malformations of PAH, most likely from mutations in the catalytic and tetramerization domains (2). Mutations in the PAH gene prevents the translated PAH from folding properly, and the unstable conformation of PAH inhibits the catabolism of L-phe. Both the primary and the tertiary structures are critical in determining the functional capabilities of PAH. Therefore, PAH can serve as a significant biomarker for PKU research as well as a general model for studying misfolded-loss-of-function proteins (1).
PAH has three domains: regulatory, catalytic, and tetramerization. The catalytic domain, consisting of residues 117 to 424, is highly conserved and is especially important for PAH’s enzymatic function. The binding sites for substrate, ligand, and cofactor are located in the catalytic domain (1-3). Most notably, catecholamine derivatives, or just simply catechols, are substrates that bind to the active site and inhibit PAH. Maintaining an adequate balance of L-phe levels is imperative because L-phe is a precursor in protein building. Therefore, inhibiting some PAH prevents complete catabolism of L-phe and allows the remaining L-phe to be incorporated into building proteins (1).
The secondary structures of PAH’s catalytic domain consist of 13 α-helices and 8 β- strands (2). The catalytic domain was co-crystallized as a complex with multiple catecholamine derivatives: dopamine, noradrenaline, L-DOPA, and adrenaline. Dopamine, noradrenaline, and adrenaline are found naturally in Homo sapiens and, therefore, can potentially inhibit PAH. The high affinity between the active site and the three catechol inhibitors leads to tight binding, whereas L-DOPA’s incompatible carboxyl chain results in less favorable binding. However, PAH-catechol complexes for all four inhibitors are structurally identical (3).
PAH crystals were analyzed using x-ray diffraction to compare catecholamine inhibition mechanisms. The success of complex formation was indicated by a blue-green hue of the crystals, which resulted from ferric iron reacting with a catechol (3). The crystallized PAH was a homodimer comprised of only the catalytic domains. However, the catalytic domain, along with the tetramerization domain, can form a tetramer by adopting alternate conformations. Both the tetramer and its components are asymmetric due to tight packing of the two domains. Each subunit has one catalytic and one tetramerization domain, and two asymmetric subunits form one tetramer. A distinctive coiled coil motif can be observed in the center of a PAH tetramer. Mammalian PAH is generally found as a tetramer. Especially in acidic environments, the equilibrium of PAH monomers shifts towards forming a tetramer. Experimentally, only a small fraction of the whole tetramer has been studied, and the complete PAH tetramer subunits have yet to be identified (1, 2).
Ferric iron ligand, which is a six-coordinate iron, is bound to the active site via three stabilizing residues: His-285, His-290, and Glu-330. The three residues firmly anchor the iron ligand in a cis orientation. The remaining three coordination sites in the opposite face are occupied by water until substrates with higher affinity are available in the solvent for coordination (4). Natural cofactors, BH4 and O2, donate electrons to reduce and activate the ferric iron ligand. The activated ligand is oxidized in the presence of catecholamines from ferrous iron (II) to ferric iron (III). When the ligand is oxidized, PAH becomes temporarily inactive. The reduction of ferric iron by natural cofactors can reverse the process (3).
Catecholamine derivatives replace two water molecules in the coordination sites by binding to the iron center via bidentate coordination. Two hydroxyl groups of catechols, one in the axial position and the other in the equatorial position, coordinate to the ferric iron ligand. Glu-330 and Tyr-325 are responsible for recognizing catechols. Hydroxyl groups of the two residues hydrogen bond with the hydroxyl groups of the catechols. The hydrogen bonds stabilize the inhibitor in the active site. In addition, Glu-330 is responsible for pH-dependent catechol binding. At neutral pH, a pKa value of 5.1 in the active site results in low dissociations of catechol inhibitors. PAH, with an isoelectric point of 6.15, has Glu-330 in the active site that can achieve the acidic pKa required for the tight binding of catechols (3, 5). Catecholamines are tightly bound to the iron center due to the partial negative charge of the carboxyl group in Glu-330 stabilizing the hydroxyl groups of the catechols at neutral pH. At low pH, the protonated carboxyl group is unable to stabilize the hydroxyl proton of the catechol. Therefore, catechol inhibitors have a higher affinity for the active site at neutral pH than at low pH (3).
PAH belongs to the aromatic amino acid hydroxylase family comprised of tryptophan hydroxylase (TRH) and tyrosine hydroxylase (TYH). The family shares most of the enzymatic functions and structures, but there is variation in affinities for certain substrates. Highly conserved genes, homologous ligands and cofactors allow for a direct comparison for substrate specificity among the three hydroxylases (2).
Tyrosine hydroxylase in rats, also known as tyrosine 3-monooxygenase of Rattus norvegicus, (PDB ID: 2TOH) shares most of its tertiary structure and biochemical function with PAH. The highly homologous sequence between the two enzymes suggests a common ancestral origin (2). The program PSI-BLAST was utilized to compare the sequence of PAH to a protein database to locate proteins with similar primary structures. E value increases as the number of gaps between the assigned protein and the aligned protein increases. E values below 0.5 indicate significant homology in the sequence. TYH has an E value of 0.0, signifying a highly unlikely alignment due to pure chance. A low E value indicates that the primary structures of PAH and of TYH are almost identical when aligned (6).
Because sequence dictates protein folding and ultimately its enzymatic function, the highly homologous primary structures of TYH and PAH explains their conserved biochemical roles. TYH executes a similar hydroxylation mechanism on the aromatic side chains of L-phe, L- tryptophan (L-trp), and L-tyr. Hydroxylation of L-try and L-tyr marks the beginning of serotonin and catecholamine neurotransmitters synthesis (7). Through DALI server, the tertiary structure of PAH was scanned against an online database to identify proteins with similar three-dimensional structures. TYH structure is superimposed on the structure of PAH and the intramolecular distances of α-carbons are compared and scored. Significantly homologous tertiary structures have a Z score above 2, and TYH has a Z score of 47.8 (8).
The three-dimensional structure of TYH greatly resembles that of PAH. TYH is found as a tetramer in nature and contains the same coiled coil motif in the center (2). The catalytic domains are conserved throughout the binding sites within the aromatic amino acid hydroxylase family. Three residues, two histidines and one glutamate, are responsible for binding ferric iron to the active site. Both PAH and TYH are nonheme iron enzymes with solvent exposed coordination sites. Furthermore, regulatory pathways for PAH and TYH are similar; both are activated by the phosphorylation of serine residues in the regulatory domain, and both are inhibited by binding of catechols in the active site. In addition, TYH serves an important biological role for neuroendocrine cells via feedback catechol inhibition by dopamine, noradrenaline, and adrenaline (3).
As similar as the two hydroxylases are functionally and structurally, TYH and PAH still have minor conformational differences. TYH is only found as a tetramer in nature, and has symmetric subunits unlike PAH. TYH’s extra hydroxyl group on the aromatic ring raises the pKa to 5.3 in the active site. Positional differences are observed in residues 245 to 250 in PAH and from 291 to 296 in TYH. This loop of residues is where the natural cofactor binds to the active site via hydrogen bonding. Glu-310 in TYH replaces His-264 in PAH. These residues stabilize the cofactor by forming hydrogen bonds between the carbonyl oxygen and the hydroxyl group of the cofactor. In addition to these positional differences, other residue variations exist in the activation sites of TYH and PAH. The combination of residue differences in the active sites determine the various substrate specificities for PAH and TYH (3).
Identifying specific substrates for PAH can serve as an invaluable tool in target drug research for PKU. A substrate with high affinity for PAH can possibly stabilize the malformed conformation and restore PAH’s enzymatic function. Future research focusing on identifying tertiary structures affected by PAH gene mutations will facilitate drug discovery process. Synthetic chaperones may be used to target specific misfoldings.