Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH).

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 (BH4) 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 BH4 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 BH4 and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH4 crosses the blood–brain barrier and therapeutic regimes based on BH4 supplementation should thus consider the effect on TH. Furthermore, BH4 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.

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum.

J. Neurochem. (2010) 114, 853–863. Abstract 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.