Serotonin (5-hydroxytryptamine, 5-HT) has a plethora of functions in both the central nervous system and the periphery. It is synthesized from tryptophan by the sequential action of two enzymes, tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase with TPH catalyzing the rate-limiting step. Two forms of TPH have been identified in mammals: TPH1, expressed highly in the gastrointestinal tract and the pineal gland, is responsible for >90% of 5-HT synthesis in the periphery, while TPH2, expressed in neuronal cells located in the dorsal raphe nucleus of the brain and the myenteric plexus of the gut, is responsible for the vast majority of 5-HT synthesis in the central nervous system [1Cote F, Thevenot E, Fligny C, et al. Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function Proc Natl Acad Sci USA 2003; 100: 13525-30.-4Liu Q, Yang Q, Sun W, et al. Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract J Pharmacol Exp Ther 2008; 325: 47-55.]. The two enzymes, sharing an overall identity of ~80% at the amino acid level, are homologous to the other two aromatic amino acid hydroxylases, phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), with approximately 50% amino acid sequence identity [3Walther DJ, Peter JU, Bashammakh S, et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform Science 2003; 299: 76., 5Flatmark T, Stevens RC. Structural insight into the aromatic amino acid hydroxylases and their disease-related mutant forms Chem Rev 1999; 99: 2137-60.]. The four enzymes use the same cofactors, tetrahydrobiopterin, iron, and oxygen to catalyze the hydroxylation of their respective substrates [6Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation Biochemistry 2003; 42: 14083-91.].

X-ray crystal structures have been determined for various forms of TPH1, PAH and TH [7Erlandsen H, Fusetti F, Martinez A, Hough E, Flatmark T, Stevens RC. Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria Nat Struct Biol 1997; 995-1000.-13Goodwill KE, Sabatier C, Marks C, Raag R, Fitzpatrick PF, Stevens RC. Crystal structure of tyrosine hydroxylase at 2.3 A and its implications for inherited neurodegenerative diseases Nat Struct Biol 1997; 4: 578-85.]. The catalytic domains of all three enzymes have a very similar fold with a mixture f α and β structures. The active site is formed by two long channels, one occupied by the amino acid substrate, the other one by pterin, with iron sitting at the intersection of the two channels. For PAH, binding of 5,6,7,8-tetrahydro-L-biopterin (BH4) caused small structural changes to the catalytic domain [8Andersen OA, Flatmark T, Hough E. High resolution crystal structures of the catalytic domain of human phenylalanine hydroxylase in its catalytically active Fe(II) form and binary complex with tetrahydrobiopterin J Mol Biol 2001; 314: 279-91.]. In contrast, binding of a phenylalanine analogue, such as L-norleucine or 3-(2-thienyl)-L-alanine (THA) in the presence of BH4 and iron ion led to large structural changes in the catalytic domain [9Andersen OA, Stokka AJ, Flatmark T, Hough E. 2.0A resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine: substrate specificity and molecular motions related to substrate binding J Mol Biol 2003; 333: 747-57., 14Andersen OA, Flatmark T, Hough E. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation J Mol Biol 2002; 320: 1095-8.]. Specifically, the loop containing residues 133-141 of PAH moved much closer to the catalytic site. For TPH1, a structure of the catalytic domain of the human enzyme with 7,8-dihydrobiopterin (BH2) and ferric iron ion bound was solved first, which showed remarkable similarity to the structure of the catalytic domain of PAH with BH4 bound [11Wang L, Erlandsen H, Haavik J, Knappskog PM, Stevens RC. Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin Biochemistry 2002; 41: 12569-74.]. More recently, a structure of the catalytic domain of chicken TPH1 with tryptophan bound was solved [12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.]. Compared with the BH2-bound structure of human TPH1, binding of tryptophan caused major structural changes with two loops (Leu124-Asp139 and Ile367-Thr369) brought closer to the catalytic site, as in the corresponding regions of PAH after substrate analogue binding [12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.].

The kinetic mechanism of mammalian TH and bacterial PAH has been determined. For both enzymes, all three substrates, i.e., amino acid, pterin, and oxygen, have to be bound before catalysis can happen [6Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation Biochemistry 2003; 42: 14083-91.]. For rat TH, the order of substrate binding is pterin first, followed by oxygen, and then tyrosine [15Fitzpatrick PF. Steady-state kinetic mechanism of rat tyrosine hydroxylase Biochemistry 1991; 30: 3658-62.]. For bacterial PAH, one study reported oxygen binding first, followed by pterin and amino acid in random order [16Pember SO, Johnson KA, Villafranca JJ, Benkovic SJ. Mechanistic studies on phenylalanine hydroxylase from Chromobacterium violaceum. Evidence for the formation of an enzyme-oxygen complex Biochemistry 1989; 28: 2124-30.]. A more recent study, however, conclusively showed that pterin binds first, followed by phenylalanine and then oxygen [17Volner A, Zoidakis J, Abu-Omar MM. Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases J Biol Inorg Chem 2003; 8: 121-8.]. Such an ordered binding mechanism with pterin binding first is consistent with the observations that both enzymes are inhibited by their amino acid substrates at high concentrations [6Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation Biochemistry 2003; 42: 14083-91.]. TPH1 is also inhibited by high concentrations of Trp [18Moran GR, Daubner SC, Fitzpatrick PF. Expression and characterization of the catalytic core of tryptophan hydroxylase J Biol Chem 1998; 273: 12259-66.], implicating a similar kinetic mechanism , even though no detailed mechanism studies have been reported.

Dysregulation of the serotonergic system in the periphery is associated with various kinds of maladies, including chemotherapy-induced nausea and vomiting [19Andrews PL, Davis CJ, Bingham S, Davidson HI, Hawthorn J, Maskell L. The abdominal visceral innervation and the emetic reflex: pathways, pharmacology, and plasticity Can J Physiol Pharmacol 1990; 68: 325-45.], gastrointestinal disorders [20Gershon MD. Nerves, reflexes, and the enteric nervous system: pathogenesis of the irritable bowel syndrome J Clin Gastroenterol 2005; 39: S184-93., 21Spiller R. Serotonin and GI clinical disorders Neuropharmacology 2008; 55: 1072-80.], and pulmonary hypertension [22Maclean MR, Dempsie Y. Serotonin and pulmonary hypertension-from bench to bedside? Curr Opin Pharmacol 2009; 9: 281-6.]. Inhibition of TPH1 will lead to decrease or total abolishment of serotonin synthesis, and thus may provide effective treatment for such disorders that are caused by excessive synthesis or release of serotonin. Previously, we reported the discovery and characterization as well as structure-activity relationship of three series of novel TPH1 inhibitors that are able to deplete peripheral but not central serotonin [4Liu Q, Yang Q, Sun W, et al. Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract J Pharmacol Exp Ther 2008; 325: 47-55., 23Shi ZC, Devasagayaraj A, Gu K, et al. Modulation of peripheral serotonin levels by novel tryptophan hydroxylase inhibitors for the potential treatment of functional gastrointestinal disorders J Med Chem 2008; 51: 3684-7., 24Jin H, Cianchetta G, Devasagayaraj A, et al. Substituted 3-(4-(1, 3, 5-triazin-2-yl) phenyl)-2-aminopropanoic Acids as Novel Tryptophan Hydroxylase Inhibitors Bioorg Med Chem 2009; 19: 5229-32.]. We have now determined X-ray structures of the catalytic domain of human TPH1 co-crystallized with one compound from each of the three series of inhibitors. Hereby, we report the structure and analysis of the co-crystals. We also carried out kinetic studies of the three inhibitors in attempt to understand their mechanism of inhibition.

We determined the crystal structures of the catalytic domain of human TPH1 in complex with three novel TPH inhibitors. The structures of TPH1 in these complexes resemble the structure of the chicken TPH1 (3E2T) co-crystallized with tryptophan and iron ion (3E2T) more than that of human TPH1 bound with BH2 and iron ion (1MLW). Superposition of all five structures shows that the enzyme adopts a more compact structure when bound with either the phenylalanine-based inhibitors or tryptophan than by the pterin analogue BH2. In all three inhibitor-TPH1 structures, the loop Ile336-Phe369 moved toward the loop Phe263-Pro266, closing around the amino acid binding pocket when compared to the TPH1-BH2-Fe(III) structure. Such a movement was also observed in the chicken TPH1-Trp-Fe(III) structure. Another large structural change observed in the chicken TPH1-Trp-Fe(III) structure is the movement of the loop Leu124-Asp139 toward the catalytic site [12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.]. Unfortunately, this loop was not resolved in any of the three inhibitor-TPH1 structures due to poor electron density. Such a movement is suggested to have also occurred in these structures, given the confirmed movement of the Ile336-Phe369 loop and the similarity in interactions between the phenylalanine moiety of the inhibitors and tryptophan with the enzyme. Since phenylalanine is also an effective substrate of TPH1 with its catalytic domain showing comparable V/K and Vmax for the two amino acids [18Moran GR, Daubner SC, Fitzpatrick PF. Expression and characterization of the catalytic core of tryptophan hydroxylase J Biol Chem 1998; 273: 12259-66.], it is not surprising that the structures of the enzyme bound with the phenylalanine-based inhibitors are more similar to the one bound with Trp.

The pattern of the iron ion coordination is different among the three complexes. Two of the structures (3HF6 and 3HFB) show a monodentate coordination by Glu317, identical to the pattern in TPH1-BH2-Fe(III) and PAH-BH4-Fe(II). In contrast, in the structure of LP-533401-TPH1 (3HF8), Glu317 has a bidentate interaction with the metal, making the coordination geometry very distorted. Such a bidentate interaction was also observed in the structures of the complexes PAH-BH4-THA-Fe(II) and TPH1-Trp-Fe(III) [9Andersen OA, Stokka AJ, Flatmark T, Hough E. 2.0A resolution crystal structures of the ternary complexes of human phenylalanine hydroxylase catalytic domain with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine or L-norleucine: substrate specificity and molecular motions related to substrate binding J Mol Biol 2003; 333: 747-57., 12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94., 14Andersen OA, Flatmark T, Hough E. Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation J Mol Biol 2002; 320: 1095-8.]. In the bidentate interaction mode, iron ion is only coordinated to five sites, allowing one open site for oxygen [12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.]. Interestingly, the mechanism of catalysis by these aromatic amino acid hydroxylases is hypothesized to have a step that involves the formation of a Fe(II)-O-O-BH4 intermediate for oxygen activation followed by the formation of Fe(IV)O for hydroxylation [6Fitzpatrick PF. Mechanism of aromatic amino acid hydroxylation Biochemistry 2003; 42: 14083-91.]. Therefore, it is tempting to suggest that of the three inhibitors, LP-533401 binds to the enzyme in a conformation that is closest to its catalytic state.

The other major difference among the three structures is the conformation of Tyr235. This residue is conserved in all known TPH1 and TPH2 sequences. In PAH and TH, this position is leucine. When Tyr235 was changed to an alanine or leucine, the mutant enzyme showed much lower specific activity (~5% remaining) and increased Km for Trp but decreased Km for BH4 [29Jiang GC, Yohrling GJT, Schmitt JD, Vrana KE. Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling J Mol Biol 2000; 302: 1005-7.], implying a role in both pterin and Trp binding. Tyr235 was indeed found to interact not only with pterin in the TPH1-BH2-Fe(III) structure but also with Trp in the TPH1-Trp-Fe(III) structure [11Wang L, Erlandsen H, Haavik J, Knappskog PM, Stevens RC. Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin Biochemistry 2002; 41: 12569-74., 12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.]. In the TPH1-LP-521834-Fe(III) structure, Tyr235 has a conformation similar to the one in Trp- or BH2-bound structure. However, in the TPH1 structures containing either LP-533401 or LP-534193, it is rotated inward by ~90^o and forms a new hydrogen bond with Pro268. These results suggest that Tyr235 has considerable conformation freedom that may be critical to the reaction mechanism of TPH1.

The results of kinetic analysis with the three inhibitors clearly showed that they are all competitive versus tryptophan but predominantly uncompetitive versus pterin. Being competitive with tryptophan is entirely consistent with the structural data that the phenylalanine moiety of the inhibitors occupies the tryptophan binding site. Being uncompetitive with pterin is also consistent with the structural data that the side chains of the three compounds are not in the space where pterin binds. More importantly, the uncompetitive behavior of the three inhibitors strongly suggests that the substrate tryptophan and the inhibitors bind only after pterin is bound [28Segel IH. Enzyme Kinetics. USA: John Wiley & Sons 1993.]. For TH and PAH, similar analyses were used to demonstrate that pterin binds before the amino acid substrate does [15Fitzpatrick PF. Steady-state kinetic mechanism of rat tyrosine hydroxylase Biochemistry 1991; 30: 3658-62., 17Volner A, Zoidakis J, Abu-Omar MM. Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases J Biol Inorg Chem 2003; 8: 121-8.]. Intriguingly, these co-crystal structures were determined in the absence of pterin, indicating that the inhibitors can bind to the enzyme before pterin does, at least at high concentrations (inhibitors were at >500 uM during crystallization). Similarly, when the crystal structure of chicken TPH1 was solved in the absence of pterin, it was also found to contain tryptophan, even though the authors suggested that tryptophan binds after biopterin during catalysis [12Windahl MS, Petersen CR, Christensen HE, Harris P. Crystal structure of tryptophan hydroxylase with bound amino acid substrate Biochemistry 2008; 47: 12087-94.]. No detailed kinetic studies were reported for TPH1 before. One study showed that the product 5-HTP inhibits TPH1 in a mechanism that is competitive versus phenylalanine but uncompetitive versus pterin [30Ichiyama A, Hasegawa H, Tohyama C, Dohmoto C, Kataoka T. Some properties of bovine pineal tryptophan hydroxylase Adv Exp Med Biol 1976; 74: 103-7.], analogous to the behavior of the three inhibitors examined here. Given its structural similarity to tryptophan, 5-HTP is expected to occupy the tryptophan site, and thus to be competitive versus trytophan but uncompetitive versus pterin. Taken together, the data showed that, similar to PAH and TH, TPH1 also follows an ordered binding mechanism with pterin binding first. Our data don’t address the binding order of tryptophan and oxygen.

In summary, we have determined co-crystal structures of the human TPH1 catalytic domain with three inhibitors. The three inhibitors all have a phenylalanine moiety which occupies the tryptophan binding site of the enzyme. All three complexes showed an overall structure that is more similar to that of chicken TPH1 bound with tryptophan rather than that of human TPH1 bound with pterin, reflecting the structural similarity between the inhibitors and tryptophan, and the major conformational change induced by the binding of the amino acid substrate and its analogue. Kinetic analysis showed that all three inhibitors are competitive versus tryptophan but largely uncompetitive with pterin, consistent with the structural data and suggestive of an ordered binding mechanism in which tryptophan must bind after pterin in order for a productive complex to form. The data shed important light on the mechanism of TPH1 catalysis and inhibition.