Abstract
Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.
Highlights
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Analytical gel filtration revealed that rchTPH2 and ND47-rchTPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of L-Phe
The dimerizing effect induced by L-Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in halflives of rchTPH2 and ND47-rchTPH2
Summary
Users may download and print one copy of any publication from the public portal for the purpose of private study or research. Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. TPH uses the cofactor Fe2+ and the cosubstrates O2 and tetrahydrobiopterin (BH4) to generate 5-hydroxytryptophan (5HTP) by hydroxylation of L-tryptophan (L-Trp). Abbreviations 5-HT, 5-hydroxytryptamin (serotonin); 5-HTP, 5-hydroxytryptophan; AAAH, aromatic amino acid hydroxylase; ACT, aspartate kinase, chorismate mutase and TyrA; BH4, tetrahydrobiopterin; chTPH2, catalytic domain of human tryptophan hydroxylase; DSC, differential scanning calorimetry; DSF, differential scanning fluorimetry; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; L-Phe, Lphenylalanine; L-Trp, L-tryptophan; MBP, maltose binding protein; PAH, phenylalanine hydroxylase; rchTPH2, regulatory and catalytic domains of human tryptophan hydroxylase 2; rnPAH, Rattus norvegicus phenylalanine hydroxylase; SEC, size exclusion chromatography; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase.
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