ABSTRACT
Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of dopamine (DA) and other catecholamines, and its dysfunction leads to DA deficiency and parkinsonisms. Inhibition by catecholamines and reactivation by S40 phosphorylation are key regulatory mechanisms of TH activity and conformational stability. We used Cryo-EM to determine the structures of full-length human TH without and with DA, and the structure of S40 phosphorylated TH, complemented with biophysical and biochemical characterizations and molecular dynamics simulations. TH presents a tetrameric structure with dimerized regulatory domains that are separated 15 Å from the catalytic domains. Upon DA binding, a 20-residue α-helix in the flexible N-terminal tail of the regulatory domain is fixed in the active site, blocking it, while S40-phosphorylation forces its egress. The structures reveal the molecular basis of the inhibitory and stabilizing effects of DA and its counteraction by S40-phosphorylation, key regulatory mechanisms for homeostasis of DA and TH.
Subject(s)
Dopamine/pharmacology , Enzyme Inhibitors/pharmacology , Tyrosine 3-Monooxygenase/antagonists & inhibitors , Tyrosine 3-Monooxygenase/chemistry , Amino Acid Sequence , Catalytic Domain , Catecholamines/metabolism , Cryoelectron Microscopy , Dopamine/chemistry , Dopamine/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Models, Molecular , Phosphorylation , Protein Binding , Protein Domains , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Tyrosine hydroxylase (TH) is the enzyme catalyzing the rate-limiting step in the synthesis of dopamine in the brain. Developing enzyme replacement therapies using TH could therefore be beneficial to patient groups with dopamine deficiency, and the use of nanocarriers that cross the blood-brain barrier seems advantageous for this purpose. Nanocarriers may also help to maintain the structure and function of TH, which is complex and unstable. Understanding how TH may interact with a nanocarrier is therefore crucial for the investigation of such therapeutic applications. This work describes the interaction of TH with porous silicon nanoparticles (pSiNPs), chosen since they have been shown to deliver other macromolecular therapeutics successfully to the brain. Size distributions obtained by dynamic light scattering show a size increase of pSiNPs upon addition of TH and the changes observed at the surface of pSiNPs by transmission electron microscopy also indicated TH binding at pH 7. As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7. However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH. TH activity was unaffected by the binding to pSiNPs most probably because the active site stays available for catalysis, in agreement with calculations of the surface electrostatic potential pointing to the most positively charged regulatory domains in the tetramer as the interacting regions. These results reveal pSiNPs as a promising delivery device of enzymatically active TH to increase local dopamine synthesis.
Subject(s)
Nanoparticles/metabolism , Silicon/metabolism , Tyrosine 3-Monooxygenase/metabolism , Humans , Porosity , Static ElectricityABSTRACT
Tyrosine hydroxylase (TH) catalyses the (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4)-dependent conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-Dopa), which is the rate-limiting step in the synthesis of dopamine and other catecholamine neurotransmitters and hormones. Dysfunctional mutant TH causes tyrosine hydroxylase deficiency (THD), characterized by symptoms ranging from mild l-Dopa responsive dystonia to severe neuropathy. THD-associated mutations often present misfolding and a propensity to aggregate, characteristics that can also be manifested by dysregulated wild-type TH. TH - and subsequently dopamine - is also reduced in Parkinson's disease (PD) due to the selective death of dopaminergic neurons. Thus, TH is a target for stabilizing small molecular weight compounds that can function as pharmacological chaperones, restoring enzyme folding and function. In this work we carried out a screening of a compound library with 1280 approved drugs and we identified levalbuterol, a beta2-adrenergic agonist that is broadly used in asthma treatment, as an interesting validated binder of human TH. Levalbuterol stabilized TH with reduced affinity compared to dopamine, the end-product and regulatory feedback inhibitor of TH, but without compromising enzymatic activity. Moreover, levalbuterol also delays the formation of TH aggregates and makes the enzyme less sensitive to dopamine, effects that could contribute to ameliorate disorders related to TH, such as THD and PD.
