The regulatory domain of human tryptophan hydroxylase 1 forms a stable dimer

Abstract

The three eukaryotic aromatic amino acid hydroxylases phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase have essentially identical catalytic domains and discrete regulatory domains. The regulatory domains of phenylalanine hydroxylase form ACT domain dimers when phenylalanine is bound to an allosteric site. In contrast the regulatory domains of tyrosine hydroxylase form a stable ACT dimer that does not bind the amino acid substrate. The regulatory domain of isoform 1 of human tryptophan hydroxylase was expressed and purified; mutagenesis of Cys64 was required to prevent formation of disulfide-linked dimers. The resulting protein behaved as a dimer upon gel filtration and in analytical ultracentrifugation. The sw value of the protein was unchanged from 2.7–35 µM, a concentration range over which the regulatory domain of phenylalanine hydroxylase forms both monomers and dimers, consistent with the regulatory domain of tryptophan hydroxylase 1 forming a stable dimer stable that does not undergo a monomer-dimer equilibrium. Addition of phenylalanine, a good substrate for the enzyme, had no effect on the sw value, consistent with there being no allosteric site for the amino acid substrate.

Structural characterization of human tryptophan hydroxylase 2 reveals L-Phe as the superior regulatory domain ligand relevant for serotonin biosynthesis

“…In the concentration range of 3-100 μM, NΔ47-TPH2-R eluted as a dimer for all protein loading concentrations and independent of the addition of L-Phe (Figure 2A-B). This is similar to the behavior of the isolated RD of TPH1, which also has been shown to exist as a dimer at protein concentrations above 2.7 μM 27 . Although L-Phe did not affect the oligomeric state of NΔ47-TPH2-R, nanoDSF measurements showed that L-Phe had a stabilizing effect on the dimeric complex with an increase in melting temperature of up to 18 °C, consistent with the presence of a binding site as previously proposed 14 (Figure 2C).…”

“…The N-tail of TPH2 is, similar to TH, longer than that of PAH. It is thus possible that residues in the end of the tail can cover the active site in a state with dimerized RDs, as also discussed by Zhang et al 27 . Although with low confidence, the Alpha-fold 2 predicts residues 1-21 of TPH2 to cover the entrance to the active site (Extended data figure 9) 38 , suggesting at least that this region has a propensity to interact with the CD.…”

One Key and Multiple Locks: Substrate Binding in Structures of Tryptophan Dioxygenases and Hydroxylases

“…TPH1 is a homo‐tetramer belonging to the family of aromatic amino acid hydroxylases. [90] It is composed of three functional domains: a N‐terminal regulatory domain being able to dimerize, [91] a central conserved catalytic domain, and a C‐terminal oligomerization domain.…”