Jeffrey McKinney

Different properties of the central and peripheral forms of human tryptophan hydroxylase

Tryptophan hydroxylase (TPH) catalyses the rate‐limiting reaction in the biosynthesis of serotonin. In humans, two different TPH genes exist, located on chromosomes 11 and 12, respectively, and encoding two enzymes (TPH1 and TPH2) with an overall sequence identity of 71%. We have expressed both enzymes as various fusion proteins in Escherichia coli and using an in vitro transcription/translation system, and compared their solubility and kinetic properties. TPH2 is more soluble than TPH1, has a higher molecular weight and different kinetic properties, including a lower catalytic efficiency towards phenylalanine than TPH1. Both enzymes are phosphorylated by cAMP‐dependent protein kinase A. TPH2 was phosphorylated at Ser19, a phosphorylation site not present in TPH1. The differences between TPH1 and TPH2 have important implications for the regulation of serotonin production in the brain and the periphery and may provide an explanation for some of the diverging results reported for TPH from different sources in the past.

Activation and stabilization of human tryptophan hydroxylase 2 by phosphorylation and 14-3-3 binding.

TPH (tryptophan hydroxylase) catalyses the rate-limiting step in the synthesis of serotonin, and exists in two isoforms: TPH1, mainly found in peripheral tissues and the pineal body, and TPH2, a neuronal form. In the present study human TPH2 was expressed in Escherichia coli and in HEK (human embryonic kidney)-293 cells and phosphorylated using several different mammalian protein kinases. TPH2 was rapidly phosphorylated to a stoichiometry of 2 mol of phosphate/mol of subunit by PKA (protein kinase A), but only to a stoichiometry of 0.2 by Ca(2+)/calmodulin dependent protein kinase II. Both kinases phosphorylated Ser(19), but PKA also phosphorylated Ser(104), as determined by MS, phosphospecific antibodies and site-directed mutagenesis of several possible phosphorylation sites, i.e. Ser(19), Ser(99), Ser(104) and Ser(306). On average, purified TPH2 WT (wild-type) was activated by 30% after PKA phosphorylation and studies of the mutant enzymes showed that enzyme activation was mainly due to phosphorylation at Ser(19). This site was phosphorylated to a stoichiometry of up to 50% in HEK-293 cells expressing TPH2, and the enzyme activity and phosphorylation stoichiometry was further increased upon treatment with forskolin. Purified PKA-phosphorylated TPH2 bound to the 14-3-3 proteins gamma, epsilon and BMH1 with high affinity, causing a further increase in enzyme stability and activity. This indicates that 14-3-3 proteins could play a role in consolidating and strengthening the effects of phosphorylation on TPH2 and that they may be important for the regulation of serotonin function in the nervous system.

Selectivity and Affinity Determinants for Ligand Binding to the Aromatic Amino Acid Hydroxylases

Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinsons disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

A loss-of-function mutation in tryptophan hydroxylase 2 segregating with attention-deficit/hyperactivity disorder

A loss-of-function mutation in tryptophan hydroxylase 2 segregating with attention-deficit/hyperactivity disorder

Functional properties of missense variants of human tryptophan hydroxylase 2.

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate‐limiting step in serotonin biosynthesis in the nervous system. Several variants of human TPH2 have been reported to be associated with a spectrum of neuropsychiatric disorders such as unipolar major depression, bipolar disorder, suicidality, and attention‐deficit/hyperactivity disorder (ADHD). We used three different expression systems: rabbit reticulocyte lysate, Escherichia coli, and human embryonic kidney cells, to identify functional effects of all human TPH2 missense variants reported to date. The properties of mutants affecting the regulatory domain, that is, p.Leu36Val, p.Leu36Pro, p.Ser41Tyr, and p.Arg55Cys, were indistinguishable from the wild‐type (WT). Moderate loss‐of‐function effects were observed for mutants in the catalytic and oligomerization domains, that is, p.Pro206Ser, p.Ala328Val, p.Arg441His, and p.Asp479Glu, which were manifested via stability and solubility effects, whereas p.Arg303Trp had severely reduced solubility and was completely inactive. All variants were tested as substrates for protein kinase A and were found to have similar phosphorylation stoichiometries. A standardized assay protocol as described here for activity and solubility screening should also be useful for determining properties of other TPH2 variants that will be discovered in the future. Hum Mutat 30:1–8, 2009.

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2.

Tryptophan hydroxylase (TPH) catalyses the rate‐limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (Vmax) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild‐type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.

Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2

J. Neurochem. (2010) 114, 853–863.

Role of PHE313/TRP326 in Determining Substrate Specificity in Tryptophan and Phenylalanine Hydroxylases

Tryptophan hydroxylase (TPH) is a tetrahydrobiopterin (BH4)- and iron-dependent enzyme that hydroxylates L-Trp to 5-hydroxy-L-Trp using molecular oxygen. This is the rate limiting step in the synthesis of serotonin. Due to the scarcity of the enzyme in animal tissues and its instability in vitro, TPH is the least characterized of the three aromatic amino acid hydroxylases and its 3D structure is still not known. Based on the high sequence identity between the three mammalian hydroxylases we have prepared a structural model for TPH (1) (Fig. 1A). We have also determined the structure of the bound conformation of L-Trp and the inactive cofactor analogue 7,8-dihydrobiopterin (BH2) complexed with a stable form of the catalytic domain of human TPH (Δ90TPH) by NMR and by molecular docking (McKinney et al., submitted; Fig. 1B). From the structure of the complex it was inferred that residue F313 (W326 in phenylalanine hydroxylase (PAH)) may have a role in substrate specificity. In this work we report the kinetic characterization of Δ90TPH and of the mutants F313/W326.

The Conformation of Tetrahydro-Biopterin Free and Bound to Aromatic Amino Acid Hydroxylases and NOS

Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH) and nitric oxide synthase (NOS) are tetrahydrobiopterin (BH4)-dependent enzymes that catalyze the hydroxylation of the respective aromatic amino acids (PAH, TH and TPH) and the synthesis of NO from arginine (NOS), using dioxygen as additional substrate. While the aromatic amino acid hydroxylases all contain a catalytic mononuclear non-heme iron which is essential for the hydroxylation, NOS contains a cytochrome P450-type heme in the oxygenase domain where NO synthesis seems to take place. We have recently studied the structure of the complex of BH2 (the oxidized analogue of BH4) and substrate with PAH by NMR and docking 1 and, in order to get further insights on the role of the iron and tetrahydropterin cofactor in the catalytic mechanism in these enzymes, we have extended these studies to BH4. Based on the distance constraints obtained by NMR complemented by distance geometry calculations, docking into the crystal structure of the enzymes and molecular dynamic simulations, we have determined the conformation of BH4 bound to each of the four enzymes.