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Recombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress-activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in V[sub max] and a decrease in K[sub m] for 6-(R)-5,6,7,8tetrahydrobiopterin (BH[sub 4]), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high-affinity binding of 14-3-3 proteins, but only the interaction of 14-3-3 with Ser19 increased the hTH1 activity. The 14-3-3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress-activated protein kinases in the regulation of catecholamine biosynthesis. [ABSTRACT FROM AUTHOR]

Tyrosine hydroxylase (TH) has been reported to require binding of 14-3-3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14-3-3 proteins BMH1/BMH2, as well as 14-3-3 ζ and a mixture of sheep brain 14-3-3 proteins. Phosphorylation of Ser31 did not result in 14-3-3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14-3-3 isoforms BMH1/BMH2 and sheep brain 14-3-3, but not for 14-3-3 ζ. On phosphorylation of both Ser19 and Ser40, binding to the 14-3-3 ζ isoform also occurred, and the binding affinity to BMH1 and sheep brain 14-3-3 increased. Both phosphoserine-specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14-3-3 proteins. This was also found when heparin was added, or after proteolytic removal of the N-terminal 37 amino acids of Ser40-phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14-3-3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14-3-3 proteins. [ABSTRACT FROM AUTHOR]

Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling., (© 2019 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Tyrosine hydroxylase (TH) is a multi-domain, homo-oligomeric enzyme that catalyses the rate-limiting step of catecholamine neurotransmitter biosynthesis. Missense variants of human TH are associated with a recessive neurometabolic disease with low levels of brain dopamine and noradrenaline, resulting in a variable clinical picture, from progressive brain encephalopathy to adolescent onset DOPA-responsive dystonia (DRD). We expressed isoform 1 of human TH (hTH1) and its dystonia-associated missense variants in E. coli, analysed their quaternary structure and thermal stability using size-exclusion chromatography, circular dichroism, multi-angle light scattering, transmission electron microscopy, small-angle X-ray scattering and assayed hydroxylase activity. Wild-type (WT) hTH1 was a mixture of enzymatically stable tetramers (85.6%) and octamers (14.4%), with little interconversion between these species. We also observed small amounts of higher order assemblies of long chains of enzyme by transmission electron microscopy. To investigate the role of molecular assemblies in the pathogenesis of DRD, we compared the structure of WT hTH1 with the DRD-associated variants R410P and D467G that are found in vicinity of the predicted subunit interfaces. In contrast to WT hTH1, R410P and D467G were mixtures of tetrameric and dimeric species. Inspection of the available structures revealed that Arg-410 and Asp-467 are important for maintaining the stability and oligomeric structure of TH. Disruption of the normal quaternary enzyme structure by missense variants is a new molecular mechanism that may explain the loss of TH enzymatic activity in DRD. Unstable missense variants could be targets for pharmacological intervention in DRD, aimed to re-establish the normal oligomeric state of TH., (© 2018 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPH1 and TPH2) are structurally and functionally related enzymes that share a number of ligands, such as amino acid substrates, pterin cofactors and inhibitors. We have recently identified four compounds (I-IV) with pharmacological chaperone effect for PAH and phenylketonuria mutants (Pey et al. (2008) J. Clin. Invest. 118, 2858-2867). We have now investigated the effect of these compounds on the brain enzymes TH and TPH2, comparative to hepatic PAH. As assayed by differential scanning fluorimetry each of the purified human PAH, TH and TPH2 was differently stabilized by the compounds and only 3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one (compound III) stabilized the three enzymes. We also investigated the effect of compounds II-IV in wild-type mice upon oral loading with 5 mg/kg/day. Significant effects were obtained by treatment with compound III - which increased total TH activity in mouse brain extracts by 100% but had no measurable effects either on TPH activity nor on monoamine neurotransmitter metabolites dopamine, dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5-hydroxyindolacetic acid - and with 5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (compound IV) - which led to a 10-30% decrease of these metabolites. Our results indicate that pharmacological chaperones aiming the stabilization of one of the aromatic amino acid hydroxylases should be tested on other members of the enzyme family. Moreover, compound III stabilizes in vitro the human TH mutant R202H, associated to autosomal recessive L-DOPA-responsive dystonia, revealing the potential of pharmacological chaperones for the treatment of disorders associated with TH misfolding.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

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 (V(max)) 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.

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.

In the presence of thiols, tyrosine hydroxylase (TH) oxidizes L-dihydroxyphenylalanine (L-DOPA) with a specific activity of up to 140 nmol min(-1) mg(-1) at 37 degrees C and pH 7.0, which is approximately 12-50% of its TH activity under similar experimental conditions. Using assay conditions that are optimal for measuring TH activity, the specific DOPA oxidase activity of human TH is similar to that of mushroom tyrosinase, but the two enzymes are clearly different in terms of substrate specificities, cofactor dependencies, and selectivity with respect to the effects of metal chelators and other inhibitors. In the presence of an excess of dithiothreitol, 2-mercaptoethanol, cysteine, or reduced glutathione, the reaction products of the two enzymes are identical and have been identified tentatively as thioether derivatives of DOPA. Theoretically, the oxidation of L-DOPA by TH may contribute to the formation of neuromelanin (pheomelanin) in catecholaminergic neurons and in the metabolism of DOPA to reactive intermediates that can react with free thiol groups in cellular proteins. The DOPA oxidase activity of TH can lead to errors in the estimation of in vivo or in vitro TH activity, and currently used assay protocols may have to be modified to avoid interference from this activity.

It has been suggested that idiopathic parkinsonism, characterized by a loss of dopaminergic neurons of the nigrostriatal pathway, is due to the intracellular generation of reactive oxygen species, generated by a nonenzymatic or enzymatic partial reduction of dioxygen. Based on in vitro studies of the iron-containing monooxygenase tyrosine hydroxylase (TH), evidence is presented that this enzyme system may also contribute to such an oxidative stress. Thus, the purified and Fe(2+)-reconstituted recombinant human enzyme shows a time- and temperature-dependent partial uncoupling of the hydroxylation of L-tyrosine with the natural cofactor (6R)-tetrahydrobiopterin, resulting in the formation of H2O2. The degree of uncoupling of the hydroxylation reaction is significantly higher when certain substrate analogues, notably the 7-substituted isomer (7-tetrahydrobiopterin) of the natural cofactor, is used. In the presence of H2O2 and Fe2+, the addition of TH increases the production of the highly reactive.OH radical, probably via a Fenton type of reaction. It is not clear whether this in vitro reaction can mediate cellular injury in vivo. However, it is known that the distribution of TH in the central and peripheral nervous system often corresponds to that of the neuronal degeneration in idiopathic parkinsonism, a finding that is compatible with a pathogenetic effect of TH.