Crystal structure and site-specific mutagenesis of pterin-bound human phenylalanine hydroxylase

The crystal structure of the dimeric catalytic domain (residues 118-424) of human PheOH (hPheOH), cocrystallized with the oxidized form of the cofacto r (7,8-dihydro-L-biopterin, BH2), has been determined at 2.0 Angstrom resol ution. The pterin binds in the second coordination sphere of the catalytic iron (the C4a atom is 6.1 Angstrom away), and interacts through several hyd rogen bonds to two water molecules coordinated to the iron, as well as to t he main chain carbonyl oxygens of Ala322, Gly247, and Leu249 and the main c hain amide of Leu249. Some important conformational changes are seen in the active site upon pterin binding, The loop between residues 245 and 250 mov es in the direction of the iron, and thus allows for several important hydr ogen bonds to the pterin ring to be formed. The pterin cofactor is in an id eal orientation for dioxygen to bind in a bridging position between the iro n and the pterin. The pterin ring forms an aromatic pi-stacking interaction with Phe254, and Tyr325 contributes to the positioning of the pterin ring and its dihydroxypropyl side chain by hydrophobic interactions. Of particul ar interest in the hPheOH . BH2 binary complex structure is the finding tha t Glu286 hydrogen bonds to one of the water molecules coordinated to the ir on as well as to a water molecule which hydrogen bonds to N3 of the pterin ring. Site-specific mutations of Glu286 (E286A and E286Q), Phe254 (F254A an d F254L), and Tr325 (Y325F) have confirmed the important contribution of Gl u286 and Phe254 to the normal positioning of the pterin cofactor and cataly tic activity of hPheOH. Tyr325 also contributes to the correct positioning of the pterin, but has no direct function in the catalytic reaction, in agr eement with the results obtained with rat TyrOH [Daubner, S. C., and Fitzpa trick, P. F. (1998) Biochemistry 37, 16440-16444]. Superposition of the bin ary hPheOH . BH2 complex onto the crystal structure of the ligand-free rat PheOH (which contains the regulatory and catalytic domains) [Kobe, B., Jenn ings, I. G,, House, C. M., Michell, B. J., Goodwill, K. E., Santarsiero, B. D., Stevens, R. C., Cotton, R. G. II., and Kemp, B. E. (1999) Nat. Struct. Biol. 6, 442-448] reveals that the C2'-hydroxyl group of BH2 is sufficient ly close to form hydrogen bonds to Ser23 in the regulatory domain. Similar interactions are seen with the hPheOH adrenaline complex and Ser23. These i nteractions suggest a structural explanation for the specific regulatory pr operties of the dihydroxypropyl side chain of BH4 (negative effector) in th e full-length enzyme in terms of phosphorylation of Ser16 and activation by L-Phe.