Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization

Abstract

Tryptophan hydroxylase 2 ( TPH 2) 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 TPH 2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH 2 (rc h TPH 2; regulatory and catalytic domain, NΔ47‐rc h TPH 2; truncation of 47 residues in the N terminus of rc h TPH 2, and c h TPH 2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. c h TPH 2 displayed 14‐ and 11‐fold higher half‐lives compared to rc h TPH 2 and NΔ47‐rc h TPH 2, 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 rc h TPH 2 and NΔ47‐rc h TPH 2 are found to shift from polyphasic to apparent two‐state by the addition of l ‐Trp or l ‐Phe. Analytical gel filtration revealed that rc h TPH 2 and NΔ47‐rc h TPH 2 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 rc h TPH 2 and NΔ47‐rc h TPH 2. Addition of l ‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of h TPH 2.

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

“…Purification. The purification procedure comprises an affinity chromatography step utilizing a column packed with dextrin sepharose and a size exclusion step using a HiLoad Superdex 200 prep grade column, as previously described 26 . All variants were cleaved from the MBP fusion protein utilizing an on-column cleavage procedure with MBP-3CP during the first purification step.…”

“…Activity Assay. The activity assay was performed as described by Moran et al 27 and Nielsen et al 25 , and the sample preparation was conducted as previously described 26 . The activity measurements were performed using a Varian Cary Eclipse Fluorescence Spectrophotometer.…”

“…The purification procedure comprises an affinity chromatography step utilizing a column packed with dextrin sepharose and a size exclusion step using a HiLoad Superdex 200 prep grade column, as previously described. 26 All variants were cleaved from the MBP fusion protein utilizing an on-column cleavage procedure with MBP-3CP during the first purification step. Human TPH1 variants were purified in buffer containing 20 mM TRIS/H 2 SO 4 , 100 mM (NH 4 ) 2 SO 4 , pH 8.0 and human TPH2 variants in 20 mM HEPES/NH 4 OH, 100 mM (NH 4 ) 2 SO 4 , pH 7.0.…”

“…The activity assay was performed as described by Moran et al 27 and Nielsen et al, 25 and the sample preparation was conducted as previously described. 26 The activity measurements were performed using a Varian Cary Eclipse fluorescence spectrophotometer. For activity measurements, the TPH variants were thawed under running water and filtered, and the concentrations were determined by UV−vis absorption at 280 nm.…”

Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli

“…Given that Tyr is an essential amino acid for E. coli propagation, a genetic selection that couples growth of a Tyr-auxotrophic strain to the catalytic activity of heterologous PheH and enzymes for the provision of tBPt can be established (Figure 2). In combination with adaptive laboratory evolution (ALE), a proven tool to improve 5), reticuline (6), epinephrine (7), physostigmine (8), serotonin (9), violacein (10), octopamine (11), and phenylephrine (12). cell robustness via growth-rate selection, 4 directed evolution of a PheH-dependent metabolic pathway in vivo becomes feasible.…”