Conformation of the substrate and pterin cofactor bound to human tryptophan hydroxylase. Important role of Phe313 in substrate specificity

ABSTRACT: Tryptophan hydroxylase (TPH) carries out the 5-hydroxylation of L-Trp, which is the ratelimiting step in the synthesis of serotonin. We have prepared and characterized a stable N-terminally truncated form of human TPH that includes the catalytic domain (∆90TPH). We have also determined the conformation and distances to the catalytic non-heme iron of both L-Trp and the tetrahydrobiopterin cofactor analogue L-erythro-7,8-dihydrobiopterin (BH 2 ) bound to ∆90TPH by using 1 H NMR spectroscopy. The bound conformers of the substrate and the pterin were then docked into the modeled three-dimensional structure of TPH. The resulting ternary TPH‚BH 2 ‚L-Trp structure is very similar to that previously determined by the same methods for the complex of phenylalanine hydroxylase (PAH) with BH 2 and L-Phe [Teigen, K., et al. (1999) J. Mol. Biol. 294, 807-823]. In the model, L-Trp binds to the enzyme through interactions with Arg257, Ser336, His272, Phe318, and Phe313, and the ring of BH 2 interacts mainly with Phe241 and Glu273. The distances between the hydroxylation sites at C5 in L-Trp and C4a in the pterin, i.e., 6.1 ( 0.4 Å, and from each of these sites to the iron, i.e., 4.1 ( 0.3 and 4.4 ( 0.3 Å, respectively, are also in agreement with the formation of a transient iron-4a-peroxytetrahydropterin in the reaction, as proposed for the other hydroxylases. The different conformation of the dihydroxypropyl chain of BH 2 in PAH and TPH seems to be related to the presence of nonconserved residues, i.e., Tyr235 and Pro238 in TPH, at the cofactor binding site. Moreover, Phe313, which seems to interact with the substrate through ring stacking, corresponds to a Trp residue in both tyrosine hydroxylase and PAH (Trp326) and appears to be an important residue for influencing the substrate specificity in this family of enzymes. We show that the W326F mutation in PAH increases the relative preference for L-Trp as the substrate, while the F313W mutation in TPH increases the preference for L-Phe, possibly by a conserved active site volume effect. Tryptophan hydroxylase (TPH) 1 is a tetrahydrobiopterinand non-heme iron-dependent enzyme that hydroxylates L-tryptophan (L-Trp) to 5-hydroxy-L-Trp using (6R)-Lerythro- These tetrameric enzymes are organized in a regulatory N-terminal domain, a catalytic domain, and a C-terminal oligomerization domain, and they exhibit extensive sequence similarity at the catalytic domains. Due to the scarcity of the enzyme in animal tissues and its instability in vitro, TPH is the least characterized enzyme of the three aromatic amino acid hydroxylases. Although significant progress has been reported recently on the structural characterization of both TH (4) and PAH (5-7), the three-dimensional (3D) structure of TPH is still not known. The difficulties encountered in the crystallization of this enzyme seem to be related to its instability and insolubility, notably when it is expressed in bacterial systems (8). However, the catalytic domain of the enzyme from different sources, including the human brain, appears to be more stable, and several groups have reported its purification and characterization (9). Recently, stable full-length TPH forms from the human pineal gland (10) and the human parasite Schistosoma mansoni (11) have been cloned, expressed, and successfully isolated. The first observable product from the pterin cofactor in the TPH reaction is a 4a-hydroxytetrahydropterin, in which the oxygen atom a