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

Abstract

The crystal structures of the catalytic domain (ΔN1-102/ΔC428-452) of human phenylalanine hydroxylase (hPheOH) in its catalytically competent Fe(II) form and binary complex with the reduced pterin cofactor 6( R)- l- erythro-5,6,7,8-tetrahydrobiopterin (BH 4) have been determined to 1.7 and 1.5 Å, respectively. When compared with the structures reported for various catalytically inactive Fe(III) forms, several important differences have been observed, notably at the active site. Thus, the non-liganded 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-spin Fe(III) form, as well as poor electron density for parts of the coordinating side-chain of Glu330. The reduced cofactor (BH 4), which adopts the expected half-semi chair conformation, is bound in the second coordination sphere of the catalytic iron with a C4a-iron distance of 5.9 Å. BH 4 binds at the same site as l- erythro-7,8-dihydrobiopterin (BH 2) in the binary hPheOH-Fe(III)·BH 2 complex forming an aromatic π-stacking interaction with Phe254 and a network of hydrogen bonds. However, compared to that structure the pterin ring is displaced about 0.5 Å and rotated about 10°, and the torsion angle between the hydroxyl groups of the cofactor in the dihydroxypropyl side-chain has changed by ∼120° enabling O2′ to make a strong hydrogen bond (2.4 Å) with the side-chain oxygen of Ser251. Carbon atoms in the dihydroxypropyl side-chain make several hydrophobic contacts with the protein. The iron is six-coordinated in the binary complex, but the overall coordination geometry is slightly different from that of the Fe(III) form. Most important was the finding that the binding of BH 4 causes the Glu330 ligand to change its coordination to the iron when comparing with non-liganded hPheOH-Fe(III) and the binary hPheOH-Fe(III)·BH 2 complex.