Serotonin is important in both physiological and mental health. Here, new insight into the structure-function relationship of the enzymetryptophan hydroxylase (TPH) involved in the biosynthesis of serotonin is presented.
Dysfunction of the level of serotonin (5-hydroxytryptamine, 5-HT) is associated with a variety of physiological and psychiatric disorders. It is thus highly relevant to investigate tryptophan hydroxylase that consists of two isoforms and catalyzes the first and rate-limiting step in the biosynthesis of serotonin.
Serotonin exerts its function by acting on several different receptors distributed throughout the entire human body. In the peripheral tissues, serotonin acts as a hormone to constrict large blood vessels and regulates platelet adhesion. Serotonin is also found in the intestines. Dysregulation of peripheral serotonin is involved in several conditions including gastro-intestinal disorders, lung fibrosis, carcinoid syndrome, and osteoporosis. In the brain, serotonin is involved in regulating centers that control wakefulness, temperature regulation, blood-pressure regulation, aggressive behavior, and sexual behavior. Disorders such as depression, schizophrenia, autism, and ADHD have been proposed to be linked to serotonin dysfunction.
TPH catalyzes the hydroxylation of tryptophan to L-5-hydroxytryptophan, which is the first and ratelimiting step in the biosynthesis of serotonin. The active form of TPH contains iron(II) and catalyze stryptophan hydroxylation utilizing 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and molecular oxygen. TPH exists in two isoforms. TPH1 is primarily expressed in the peripheral tissues, and TPH2 in the central nervous system. Both isoforms are members of an enzyme family of iron(II)-containing monooxygenases.
Of the two isoforms, TPH2 is less characterized in literature due to its inherent instability. To overcome this challenge, three variants of human TPH2 were expressed, purified, and examined. Removal of the C-terminal tetramerization domain resulted in sufficient quantities for characterization. Upon further removal of the N-terminal regulatory domain, a significant decrease in rate of inactivation was observed. This observation renders the regulatory domain the main source of instability. To overcome the inherent instability of the regulatory domain, differential scanning fluorimetry was used to identify stabilizing ligands. Analytical gel filtration revealed that in the presence of the regulatory domain, the TPH2 variant resides in a monomer-dimer equilibrium. With the addition of phenylalanine, a significant shift towards dimer was observed explaining the ligand-induced increase in thermo-stability. These results led to the addition of phenylalanine in the purification buffer which significantly increase the purification yields.
Further results demonstrate, that the steady-state kinetic mechanism of the catalytic domain of human TPH1 follows a hybrid Ping Pong ordered mechanism. The kinetic study also revealed that the isoforms display very different kinetic properties despite their high sequence identity. TPH1 is substrate inhibited, while TPH2 is not. By scrutinizing the crystal structures of the isoforms, it was found that differences reside in the orientation of a loop lining the active site. Point mutations were conducted within this loop, and significant changes in the kinetic parameters of the mutant TPH1 variants were observed.
Molecular dynamics simulations revealed that the substrate inhibition mechanism occurs through a closure of the BH4 binding pocket upon tryptophan binding, and that the active site loop is involved in this mechanism by propagating structural changes from the tryptophan binding site to the BH4 binding pocket.
Crystal structure of human chTPH1 with BH2 and iron (PDB entry: 1MLW) and L-tryptophan (superimposed from chicken TPH1, PDB entry: 3E2T [244]).