Journal of Inorganic Biochemistry NON-HEME IRON 313

M02 CRYSTAL STRUCTURE OF TYROSINE AND PHENYLALANINE HYDROXYLASE CATALYTIC DOMAIN

lL~_.~.tg..~d~, K.G. Goodwill, C. Sabatier, and F. Fussetti. Department of Chemistry, University of California, Berkeley, CA 94720, USA

The aromatic amino acid hydroxylase family of enzymes is composed of tyrosine hydroxylase, phenylalanine hydroxylase, and tryptophan hydroxylase. This family of enzymes share a common catalytic mechanism, utilizing an aromatic amino acid and molecular oxygen as substrates, tetrahydrobiopterin as a cofactor, and a ferrous iron atom. Tyrosine hydroxylase catalyzes the conversion of tyrosine to L-DOPA, the rate limiting step in catecholamine biosynthesis. Phenylalanine hydroxylase catalyzes the catabolism of phenylalanine, and tryptophan hydroxylase catalyzes the conversion of tryptophan to 5-hydroxytryptophan, the rate limiting step in serotonin biosynthesis. To elucidate the catalytic mechanism of this family, structural studies have been initiated.

The three-dimensional crystal structure of the tyrosine hydroxylase catalytic domain has been determined at 2.3 A resolution. The molecular architecture displays a novel alpha-helical catalytic core basket with an open cavity forming a 17 A deep active site pocket. The catalytic iron is located 10/~ below the enzyme surface and is coordinated by .the conserved residues His 331, His 336, and Glu 376. The catalytic core is connected to a 40 A long helix involved in an anti- parallel coiled-coil tetramerization sub-domain. This structure provides a rationale for the effect of point mutations in tyrosine hydroxylase that cause L-DOPA responsive paorkinsonism and Segawa's syndrome. The pterin co-factor has been located approximatel), 5 A from the iron binding site, with the closest contact between pterin and iron being 3.5 A. The hydroxylation target on the pterin molecule is 5.5 A from the iron site, in a suitable position for dioxygen to bridge between the pterin and iron atom. Data have been collected on the apo-enzyme, pterin+enzyme complex, dopamine+enzyme complex, pterin+Tyr+iron+enzyme complex, and 3- Iodo-Tyr+enzyme complex.

The three-dimensional structure of the phenylalanine hydroxylase catalytic domain has been determined at 3.0 A resolution using the structure of tyrosine hydroxylase as a molecular replacement model. Although the resolution of the phenylalanine hydroxylase structure is not sufficient to describe detailed interactions, the resolution is sufficient to illustrate several important differences between the catalytic domains of tyrosine and phenylalanine hydroxylase. In particular, the tetramerization domain and the active site are significantly different. Mapping of 112 different point mutations observed in individuals with the disease phenylketonuria (PKU) provides a structural rationale for mutagenesis effects on the three-dimensional structure of phenylalanine hydroxylase.