Tryptophan hydroxylase 1 (TPH1) and tryptophan hydroxylase 2 (TPH2) are the enzymes catalyzing the rate-limiting step in the biosynthesis of serotonin in the peripheral tissues and in the brain, respectively. Serotonin imbalances are implicated in various diseases, of which one example is depression. Therefore, TPH1 and TPH2 are important enzymes to characterize. However, the study of the enzymes’ structure-function relationships has historically been hampered by their low stability. Notably, no structures of the regulatory domains are available in the protein data bank at the time of writing. The current work concerns characterization of both human TPH1 and human TPH2, and focuses on the binding of a suspected allosteric ligand L-phenylalanine (L-Phe) to the regulatory domains of the two enzymes.
Nuclear magnetic resonance (NMR) spectroscopy is used to determine a structure of the isolated regulatory domain of TPH2 in complex with L-Phe. It is additionally shown that TPH2’s natural substrate, L-Trp, binds considerably worse than L-Phe to the isolated regulatory domain.
Cryo-electron microscopy is used to obtain a low-resolution structure of a TPH2 tetramer containing all three domains of the enzyme, and to provide information on the conformations of the regulatory domains in the tetrameric enzyme in absence of L-Phe.
A combination of size exclusion chromatography coupled to multi-angle light scattering and NMR spectroscopy shows that the regulatory domain of TPH1 also has the ability to bind L-Phe similar to TPH2. This reveals that the regulatory domain binding pockets are not isoform specific.
The results will be valuable for future studies on the regulation of TPH1 and TPH2 activity relevant for the understanding of serotonin related diseases, and has potential to aid in future drug discovery studies.