Mapping L-phenylalanine binding to the regulatory domains of human tryptophan hydroxylase isoforms

Tryptophan hydroxylase (TPH) catalyzes the reaction of L-Tryptophan into L-5-hydroxytryptophan, which is the rate-limiting step in the biosynthesis of serotonin. In the peripheral tissues, L-Trp hydroxylation is catalyzed by tryptophan hydroxylase isoform 1 (TPH1), while tryptophan hydroxylase isoform 2 (TPH2) catalyzes the reaction in the brain. The homologous homo-tetrameric enzymes contain three domains: A regulatory (RD), a catalytic (CD), and a tetramerization domain (TD). As a hormone and neurotransmitter, serotonin influences a variety of body functions and behaviors e.g. liver function, gastrointestinal motility, appetite, aggression, and attention, and consequently dysregulation of the serotonin system is associated with several diseases. Therefore, to shed further light on serotonin regulation and serotonin related diseases, a detailed understanding of TPH’s regulation is important. In this project, the human TPH isoforms have been investigated in two different studies.
The first study concerned a structural characterization of TPH2. L-Phe has previously been shown to increase stability and induce dimerization of TPH2 variants containing the RD, which has been connected to a potential binding site in the RD. In this study, the structure of an N-terminally truncated variant of the isolated RD in complex with L-Phe was obtained using nuclear magnetic resonance (NMR) spectroscopy. L-Phe was bound at the dimer interface in two identical binding pockets, as also seen for a homologous enzyme, phenylalanine hydroxylase. It was additionally observed using NMR spectroscopy that L-Phe was superior to the natural substrate, L-Trp, as a ligand of the isolated RD. Cryo-electron microscopy was used to study an N-terminally truncated variant of the complete tetrameric enzyme, which resulted in a low-resolution structure of the variant with dimerized RDs. 2D class averages additionally indicated that the RDs exist in a monomer-dimer equilibrium in tetrameric TPH2, where RD monomers move randomly relative to the CDs.
Assessing the residues involved in L-Phe binding to the RD of TPH2, and comparing the sequences of TPH1 and TPH2, showed that many residues from the binding pockets were conserved in TPH1. The second study therefore concerned ligand binding to the RD of TPH1. Size exclusion chromatography coupled to multi-angle light scattering was used to investigate the oligomeric state of different TPH1 variants without the TD. The results showed that variants containing the RD, formed dimers and that dimer formation was stabilized by the presence of L-Phe, but not L-Trp. Additionally, NMR spectroscopy showed that the RD dimer of TPH1 bound L-Phe.