High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin

The crystal structures of the catalytic domain (Delta N1-102/Delta C428-452 ) of human phenylalanine hydroxylase (hPheOH) in its catalytically competen t Fe(II) form and binary complex with the reduced pterin cofactor 6(R)-L-er ythro-5,6,7,8-tetrahydrobiopterin (BH4) have been determined to 1.7 and 1.5 Angstrom, respectively. When compared with the structures reported for var ious catalytically inactive Fe(III) forms, several important differences ha ve been observed, notably at the active site. Thus, the nonliganded hPheOH- Fe(II) structure revealed well defined electron density for only one of the three water molecules reported to be coordinated to the iron in the high-s pin Fe(III) form, as well as poor electron density for parts of the coordin ating side-chain of Glu330. The reduced cofactor (BH4), which adopts the ex pected half-semi chair conformation, is bound, in the second coordination s phere of the catalytic iron with a C4a-iron distance of 5.9 Angstrom. BH4 b inds at the same site as L-erythro-7,8-dihydrobiopterin (BH2) in the binary hPheOH-Fe(III) . BH2 complex forming an aromatic pi -stacking interaction with Phe254 and a network of hydrogen bonds. However, compared to that stru cture the pterin ring is displaced about 0.5 Angstrom and rotated about 10 degrees, and the torsion angle between the hydroxyl groups of the cofactor in the dihydroxypropyl side-chain has changed by similar to 120 degrees ena bling O2' to make a strong hydrogen bond (2.4 Angstrom) with the side-chain oxygen of Ser251. Carbon atoms in the dihydroxypropyl side-chain make seve ral hydrophobic contacts with the protein. The iron is six-coordinated in t he binary complex, but the overall coordination geometry is slightly differ ent from that of the Fe(III) form. Most important was the finding that the binding of BH4 causes the Glu330 ligand to change its coordination to the i ron when comparing with nonliganded hPheOH-Fe(III) and the binary hPheOH-Fe (III) . BH2 complex. (C) 2001 Academic Press.