A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Nielsen, M.; Petersen, Charlotte Rode; Munch, Astrid; Vendelboe, Trine V.; Boesen, Jane; Harris, Pernille; Christensen, Hans Erik Mølager

doi:10.1016/j.pep.2007.10.016

Cited by 8 publications

(13 citation statements)

References 49 publications

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“…All DNA sequences were verified by sequencing (Eurofins). Proteins were expressed at 20 °C for 14 h, as previously described . MBP‐3CP was cloned and expressed in‐house in a similar manner.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2016

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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.

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Section: Methodsmentioning

confidence: 99%

“…Various truncated variants of all three AAAHs have been structurally characterized . For h TPH, the catalytic cores of both isoforms have been structurally and enzymatically characterized due to successful purification strategies .…”

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confidence: 99%

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

et al. 2016

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“…The recombinant fusion proteins contain a cleavage recognition site for human rhinovirus 3C protease (3CP) . Proteins were expressed at 20 °C for 14 h as previously described . MBP-3CP was cloned, expressed, and purified in-house.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: Cloning and Expressionmentioning

confidence: 99%

“…TPH activity was assayed in a reaction mixture (10x10 mm QS quartz cuvette from Hellma -2500 µL total volume) containing achieved by equilibrating the solution with a mixture of O2 and N2 as previously described. 25 Excitation and emission wavelengths were 300 and 330 nm, respectively. Initial velocity was determined from linear regression through the first 0.04 min after BH4 addition.…”

Section: Cloning and Expressionmentioning

confidence: 99%

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Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

et al. 2017

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Tryptophan hydroxylase (TPH) catalyzes the initial and rate-limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression and irritable bowel syndrome. TPH exists in two isoforms: TPH1 and TPH2. TPH1 catalyzes the initial step in the synthesis of serotonin in the peripheral tissues, while TPH2 catalyzes this step in the brain. In this study, the steady-state kinetic mechanism for the catalytic domain of human TPH1 has been determined. Varying substrate tryptophan (Trp) and tetrahydrobiopterin (BH) results in a hybrid Ping Pong-ordered mechanism in which the reaction can either occur through a Ping Pong or a sequential mechanism depending on the concentration of tryptophan. The catalytic domain of TPH1 shares a sequence identity of 81% with TPH2. Despite the high sequence identity, differences in the kinetic parameters of the isoforms have been identified; i.e., only TPH1 displays substrate tryptophan inhibition. This study demonstrates that the difference can be traced to an active site loop which displays different properties in the TPH isoforms. Steady-state kinetic results of the isoforms, and variants with point mutations in a loop lining the active site, show that the kinetic parameters of only TPH1 are significantly changed upon mutations. Mutations in the active site loop of TPH1 result in an increase in the substrate inhibition constant, K, and therefore turnover rate. Molecular dynamics simulations reveal that this substrate inhibition mechanism occurs through a closure of the cosubstrate, BH, binding pocket, which is induced by Trp binding.

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Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway

Fang

Wang

et al. 2020

Appl Microbiol Biotechnol

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A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Cited by 8 publications

References 49 publications

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

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

Isoform-Specific Substrate Inhibition Mechanism of Human Tryptophan Hydroxylase

Enhanced production of 5-hydroxytryptophan through the regulation of L-tryptophan biosynthetic pathway

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