Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis.

Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow ( kobs = 0.13 h-1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed CuII active site is inconsistent with a biogenesis pathway that proceeds through a CuI-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of 3O2 with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.

Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants.

Betalains are tyrosine-derived red-violet and yellow pigments, found in plants only of the Caryophyllales order. Although much progress has been made in recent years in the understanding of the betalain biosynthetic process, many questions remain open with regards to several of the proposed steps in the pathway. Most conspicuous by its absence is the characterization of the first committed step in the pathway, namely the 3-hydroxylation of tyrosine to form l-3,4-dihydroxyphenylalanine (l-DOPA). We used transcriptome analysis of the betalain-producing plants red beet (Beta vulgaris) and four o'clocks (Mirabilis jalapa) to identify a novel, betalain-related cytochrome P450-type gene, CYP76AD6, and carried out gene silencing and recombinant expression assays in Nicotiana benthamiana and yeast cells to examine its functionality. l-DOPA formation in red beet was found to be redundantly catalyzed by CYP76AD6 together with a known betalain-related enzyme, CYP76AD1, which was previously thought to only catalyze a succeeding step in the pathway. While CYP76AD1 catalyzes both l-DOPA formation and its subsequent conversion to cyclo-DOPA, CYP76AD6 uniquely exhibits only tyrosine hydroxylase activity. The new findings enabled us to metabolically engineer entirely red-pigmented tobacco plants through heterologous expression of three genes taking part in the fully decoded betalain biosynthetic pathway.

Controlled Striatal DOPA Production From a Gene Delivery System in a Rodent Model of Parkinson's Disease.

Conventional symptomatic treatment for Parkinson's disease (PD) with long-term L-3,4-dihydroxyphenylalanine (DOPA) is complicated with development of drug-induced side effects. In vivo viral vector-mediated gene expression encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) provides a drug delivery strategy of DOPA with distinct advantages over pharmacotherapy. Since the brain alterations made with current gene transfer techniques are irreversible, the therapeutic approaches taken to the clinic should preferably be controllable to match the needs of each individual during the course of their disease. We used a recently described tunable gene expression system based on the use of destabilized dihydrofolate reductase (DD) and generated a N-terminally coupled GCH1 enzyme (DD-GCH1) while the TH enzyme was constitutively expressed, packaged in adeno-associated viral (AAV) vectors. Expression of DD-GCH1 was regulated by the activating ligand trimethoprim (TMP) that crosses the blood-brain barrier. We show that the resulting intervention provides a TMP-dose-dependent regulation of DOPA synthesis that is closely linked to the magnitude of functional effects. Our data constitutes the first proof of principle for controlled reconstitution of dopamine capacity in the brain and suggests that such next-generation gene therapy strategies are now mature for preclinical development toward use in patients with PD.

From tyrosine to melanin: Signaling pathways and factors regulating melanogenesis.

Melanins are natural pigments of skin, hair and eyes and can be classified into two main types: brown to black eumelanin and yellow to reddish-brown pheomelanin. Biosynthesis of melanins takes place in melanosomes, which are specialized cytoplasmic organelles of melanocytes - dendritic cells located in the basal layer of the epidermis, uveal tract of the eye, hair follicles, as well as in the inner ear, central nervous system and heart. Melanogenesis is a multistep process and begins with the conversion of amino acid L-tyrosine to DOPAquinone. The addition of cysteine or glutathione to DOPAquinone leads to the intermediates formation, followed by subsequent transformations and polymerization to the final product, pheomelanin. In the absence of thiol compounds DOPAquinone undergoes an intramolecular cyclization and oxidation to form DOPAchrome, which is then converted to 5,6-dihydroksyindole (DHI) or 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Eumelanin is formed by polymerization of DHI and DHICA and their quinones. Regulation of melanogenesis is achieved by physical and biochemical factors. The article presents the intracellular signaling pathways: cAMP/PKA/CREB/MITF cascade, MAP kinases cascade, PLC/DAG/PKCß cascade and NO/cGMP/PKG cascade, which are involved in the regulation of expression and activity of the melanogenesis-related proteins by ultraviolet radiation and endogenous agents (cytokines, hormones). Activity of the key melanogenic enzyme, tyrosinase, is also affected by pH and temperature. Many pharmacologically active substances are able to inhibit or stimulate melanin biosynthesis, as evidenced by in vitro studies on cultured pigment cells.

Design of a single AAV vector for coexpression of TH and GCH1 to establish continuous DOPA synthesis in a rat model of Parkinson's disease.

Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolase-1 (GCH1), needed for DOPA production. Here, we present a novel vector design that enables efficient DOPA production from a single AAV vector in rats with complete unilateral dopamine (DA) lesions. Functional efficacy was assessed with drug-induced and spontaneous motor behavioral tests where vector-treated animals showed near complete and stable recovery within 1 month. Recovery of motor function was associated with restoration of extracellular DA levels as assessed by online microdialysis. Histological analysis showed robust transgene expression not only in the striatum but also in overlying cortical areas. In globus pallidus, we noted loss of NeuN staining, which might be due to different sensitivity in neuronal populations to transgene expression. Taken together, we present a single AAV vector design that result in efficient DOPA production and wide-spread transduction. This is a favorable starting point for continued translation toward a therapeutic application, although future studies need to carefully review target region, vector spread and dilution with this approach.

Key factors determining the efficacy of gene therapy for continuous DOPA delivery in the Parkinsonian brain.

L-DOPA is currently the standard treatment for alleviating the motor symptoms in Parkinson's disease. The therapeutic efficacy, however, diminishes as the disease progresses. It has been suggested that the beneficial effect of L-DOPA could be reestablished by changing the mode of administration. Indeed, continuous delivery of l-DOPA has been shown to be an effective way to circumvent many of the side effects seen with traditional oral administration, which results in an intermittent supply of the dopamine precursor to the brain. However, all currently tested continuous dopaminergic stimulation approaches rely on peripheral administration. This is not ideal since it gives rise to off target effects and is difficult to maintain long-term. Thus, there is an unmet need for an effective continuous administration method with an acceptable side effect profile. Viral-mediated gene therapy is a promising alternative paradigm that can meet this demand. Encouraging preclinical studies in animal models of Parkinson's disease showed therapeutic efficacy after expression of the genes encoding the enzymes required for biosynthesis of dopamine. Although the first phase I clinical trials using these approaches have been conducted, clear positive data in placebo controlled efficacy studies is still lacking. We are now at a critical junction and need to carefully review the preclinical data from the clinical translation perspective and identify the key factors that will determine the potential for success in gene therapy for Parkinson's disease.

Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein.

Loss of substantia nigra dopaminergic neurons, which develop from the ventral region of the midbrain, is associated with Parkinson's disease. During embryogenesis, induction of these and other ventral neurons is influenced by interactions with the induction of mesoderm of the notochord and the floor plate, which lies at the ventral midline of the developing CNS. Sonic hedgehog encodes a secreted peptide, which is expressed in notochord and floor plate cells and can induce appropriate ventral cell types in the basal forebrain and spinal cord. Here we demonstrate that Sonic hedgehog is sufficient to induce dopaminergic and other neuronal phenotypes in chick mesencephalic explants in vitro. We find that Sonic hedgehog is a general ventralizing signal in the CNS, the specific response being determined by the receiving cells. These results suggest that Sonic hedgehog may have utility in the induction of clinically important cell types.

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine.

The pancreatic ß cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet ß cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet ß cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet ß cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.

Gene therapy in hemiparkinsonian rhesus monkeys: long-term survival and behavioral recovery by transplantation of autologous human tyrosine hydroxylase-expressing neural stem cells.

BACKGROUND AIMS: Neural stem cells (NSC) derived from bone marrow stromal cells (BMSC) (BMSC-D-NSC) are remarkably versatile in response to environmental signals, which render them useful in the search for neurodegenerative disease treatments. METHODS: We isolated NSC from rhesus monkey bone marrow (BM), transfected them with the human tyrosine hydroxylase (hTH) gene, and transplanted them into 1-methyl-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned hemiparkinsonian rhesus monkeys to determine changes in neural transmitter production and alterations in behavior. RESULTS: hTH-expressing cells produced monoamine agents in vitro, such as noradrenalin and dopamine. After cell transplantation in the caudate nucleus and substantia nigra of the experimental monkeys, their disease symptoms and dysfunctional glucose metabolism and dopamine transport were ameliorated. CONCLUSIONS: hTH-expressing BMSC-D-NSC survived in transplantation sites and assumed normal dopaminergic neuronal properties, playing an instrumental role in functional restoration.

Catecholamine biosynthesis in brains of rats treated with morphine.

In the brains of rats without tolerance to morphine, the accumulation of [(14)C]dopamine formed from [(14)C]tyrosine injected intracisternally is increased, reaching a maximum in the hypothalamus and striatum 1 hour after administration of morphine. In tolerant rats, the rate of incorporation of carbon-14 into dopamine and into norepinephrine in these areas is more than twice that in animals that have received only one injection of morphine.

Brain catechol synthesis: control by train tyrosine concentration.

Brain catechol synthesis was estimated by measuring the rate at which brain dopa levels rose following decarboxylase inhibition. Dopa accumulation was accelerated by tyrosine administration, and decreased by treatments that lowered brain tyrosine concentrations (for example, intraperitoneal tryptophan, leucine, or parachlorophenylalanine). A low dose of phenylalanine elevated brain tyrosine without accelerating dopa synthesis. Our findings raise the possibility that nutritional and endocrine factors might influence brain catecholamine synthesis by controlling the availability of tyrosine.

Chronic treatment and withdrawal of the cannabinoid agonist WIN 55,212-2 modulate the sensitivity of presynaptic receptors involved in the regulation of monoamine syntheses in rat brain.

Brain monoamines are involved in many neurochemical and behavioral effects of cannabinoids, but little is known on the regulation of noradrenaline, dopamine, and serotonin (5-HT) synthesis in cannabinoid addiction. This study investigated in rat brain the chronic effects of the potent cannabinoid agonist WIN 55,212-2 and of rimonabant-precipitated withdrawal, as well as the sensitivity of synthesis-modulating inhibitory receptors, on the accumulation of L-3,4-dihydroxyphenylalanine (DOPA) and 5-HTP after decarboxylase inhibition. Acute WIN (8 mg/kg; 1 h) increased DOPA synthesis in cortex (52%), hippocampus (51%), and cerebellum (56%) and decreased DOPA accumulation in striatum (31%). Acute WIN also decreased the synthesis of 5-HTP in all brain regions (40-53%). Chronic WIN (2-8 mg/kg; 5 days) and/or antagonist-precipitated withdrawal induced tolerance to the acute effects of WIN on the accumulation of DOPA (cortex and striatum) and 5-HTP (all brain regions). The inhibitory effect of clonidine (alpha2-agonist; 1 mg/kg) on the accumulation of DOPA (15-41%) and 5-HTP (22-41%) was markedly decreased or abolished after chronic WIN and precipitated withdrawal, mainly in noradrenergic and serotonergic brain regions, which indicated desensitization of alpha2-autoreceptors and alpha2-heteroreceptors regulating the synthesis of noradrenaline and 5-HT. In WIN-dependent rats (chronic and withdrawal states), the effect of a low dose of (+/-)-8-hydroxy-2-(di-n-propylamino)-tetralin (5-HT1A agonist; 0.1 mg/kg) on the accumulation of precursor amino acids was markedly potentiated in cerebellum and striatum, indicating the induction of supersensitivity of 5-HT1A-autoreceptors and 5-HT1A-heteroreceptors that regulate the synthesis of 5-HT, noradrenaline, and dopamine in these brain regions. These chronic adaptations in presynaptic receptor function could play a relevant role in cannabinoid addiction.

[Amine neuromediators, their precursors, and oxidation products in the culture of Escherichia coli K-12].

Growth dynamics of the synthesis of monoamine neuromediators serotonin, norepinephrine, and dopamine in Escherichia coli K-12 was investigated for the first time using high performance liquid chromatography with electrodetection. Maximum (micromolar) concentrations of these compounds were detected in E. coli cells during the early growth phases; their intracellular content decreases after the transition to late growth phases. E. coli biomass contains the substances DOPA and 5-hydroxytryptamine that serve in animal cells as neuromediator precursors and the products of their oxidative deamination. Presumably, the biosynthesis and degradation of monoamine neuromediators in bacterial cells involves enzyme systems analogous to those typical of animals. The culture fluid of E. coli contains micromolar concentrations of DOPA and nanomolar of serotonin, dopamine, and norepinephrine during the late growth phase. These concentrations are sufficient for animal/human receptors to bind them. This article deals with the potential biotechnological applications of the data obtained.

Characterization of tyrosine hydroxylase from Manduca sexta.

In insects, 3,4-dihydroxyphenylalanine (DOPA) is required for tanning of newly formed cuticle and the production of melanin during some types of immune responses. DOPA is produced by the hydroxylation of tyrosine, and this reaction can be catalyzed by two types of enzymes: tyrosine hydroxylase (TH) and phenoloxidase (PO). TH is required for cuticle tanning in Drosophila melanogaster and for cuticle pigmentation in other insect species, but additional functions of TH have been uncertain. In contrast, an immune function for PO has been well documented. The goal of this study was to characterize TH from Manduca sexta with a focus on its possible contribution to cuticle tanning and immune-associated melanization. We cloned a full-length TH cDNA, purified recombinant TH, and confirmed that MsTH and MsPO have tyrosine hydroxylating activity. To determine possible functions, we analyzed TH expression profiles. TH mRNA and protein were present in eggs at the stage when the pharate larval cuticle begins to tan and also in the integument of molting larvae. The amount of TH in the integument was correlated with the degree of cuticle tanning. Unlike PO, which was found to be constitutively expressed by hemocytes and was present in plasma, TH was upregulated in hemocytes and the fat body in response to an immune challenge and remained intracellular. These data suggest that TH is required for cuticle tanning and immunity in M. sexta. Based on the collective information from many studies, we propose a model in which TH is a major producer of the DOPA required for both cuticle tanning and immune-associated melanization.

Copper amine oxidase: cunning cofactor and controversial copper.

Copper amine oxidases have a complex reaction cycle that converts a primary amine and molecular oxygen into the aldehyde, ammonia and hydrogen peroxide. Coupling structural studies of freeze-trapped reaction intermediates in crystals with kinetic and spectroscopic experiments in solution has generated a detailed molecular picture of catalysis. Although dioxygen has been directly observed bound to the copper at a late stage in the reaction cycle, whether copper is the initial binding site remains controversial.

A novel photoswitchable enzyme cascade for powerful signal amplification in versatile bioassays.

This report outlines the construction of an advanced, exquisite photoswitchable enzyme cascade on the basis that tyrosinase (TYR) catalyzes the generation of dihydroxyphenylalanine (DOPA) coordinated TiO2 nanoparticles (NPs) to form a light responsive nano-trigger that subsequently photoactivates the enzymatic activity of horseradish peroxidase (HRP). This photoswitchable enzyme cascade has a powerful signal transduction/amplification ability in TYR-based bioassays, and holds great promise to be applied in versatile applications.

A derivative of the glycopeptide A40926 produced by inactivation of the beta-hydroxylase gene in Nonomuraea sp. ATCC39727.

The actinomycete Nonomuraea sp. ATCC39727 produces the glycopeptide A40926. In the corresponding dbv cluster, ORF28 encodes a putative hydroxylase. A gene replacement mutant of ORF28 in Nonomuraea produces a small amount of an A40926-related metabolite, 16 amu smaller than the parent compound, which was identified as the desoxyderivative of A40926 lacking the beta-hydroxyl group on the tyrosine moiety. This result demonstrates that ORF28 is actually involved in the formation of the beta-hydroxytyrosine residue present in A40926. The formation of an altered glycopeptide and the inability to rescue A40926 production upon feeding free beta-hydroxytyrosine are consistent with the possibility that, in contrast to balhimycin formation, hydroxylation occurs after tyrosine activation by the nonribosomal peptide synthetase.

Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes.

The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.