title: The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism.
abstract: Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin and non-heme iron-dependent enzyme that hydroxylates L-Phe to l-Tyr using molecular oxygen as additional substrate. A dysfunction of this enzyme leads to phenylketonuria (PKU). The conformation and distances to the catalytic iron of both L-Phe and the cofactor analogue L-erythro-7,8-dihydrobiopterin (BH2) simultaneously bound to recombinant human PAH have been estimated by (1)H NMR. The resulting bound conformers of both ligands have been fitted into the crystal structure of the catalytic domain by molecular docking. In the docked structure L-Phe binds to the enzyme through interactions with Arg270, Ser349 and Trp326. The mode of coordination of Glu330 to the iron moiety seems to determine the amino acid substrate specificity in PAH and in the homologous enzyme tyrosine hydroxylase. The pterin ring of BH2 pi-stacks with Phe254, and the N3 and the amine group at C2 hydrogen bond with the carboxylic group of Glu286. The ring also establishes specific contacts with His264 and Leu249. The distance between the O4 atom of BH2 and the iron (2.6(+/-0.3) A) is compatible with coordination, a finding that is important for the understanding of the mechanism of the enzyme. The hydroxyl groups in the side-chain at C6 hydrogen bond with the carbonyl group of Ala322 and the hydroxyl group of Ser251, an interaction that seems to have implications for the regulation of the enzyme by substrate and cofactor. Some frequent mutations causing PKU are located at residues involved in substrate and cofactor binding. The sites for hydroxylation, C4 in L-Phe and C4a in the pterin are located at a distance of 4.2 and 4.3 A from the iron moiety, respectively, and at 6.3 A from each other. These distances are adequate for the intercalation of iron-coordinated molecular oxygen, in agreement with a mechanistic role of the iron moiety both in the binding and activation of dioxygen and in the hydroxylation reaction.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Bergen, Arstadveien 19, Bergen, N-5009, Norway.
authors.forename: K
authors.initials: K
authors.lastname: Teigen
authors.forename: N A
authors.initials: NA
authors.lastname: Frøystein
authors.forename: A
authors.initials: A
authors.lastname: Martínez
mesh_terms: D001896:Boron Compounds / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D005290:Ferric Compounds / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism*; D010651:Phenylalanine Hydroxylase / Q000378:metabolism*; D011485:Protein Binding; D011522:Protons; D011622:Pterins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012996:Solutions; D013379:Substrate Specificity
chemical_list: D001896:Boron Compounds; D005290:Ferric Compounds; D011522:Protons; D011622:Pterins; D011994:Recombinant Proteins; D012996:Solutions; D010649:Phenylalanine; D010651:Phenylalanine Hydroxylase