Biosynthesis of 3,4-dihydroxyphenylalanine in Vicia faba

Radioactive shikimic acid and l-tyrosine were shown to be efficient precursors of 3,4-dihydroxyphenylalanine (DOPA) in Vicia faba. [1-¹⁴C]Acetate and l[U-¹⁴C]phenylalanine were not incorporated into tyrosine or DOPA. Thus the synthesis of DOPA occurs via the shikimic acid pathway and tyrosine or a very closely related metabolise. Phenolase was present in etiolated plants in much larger quantities after a brief light exposure whereas DOPA concentration was relatively constant during all stages of plant growth. Partially purified phenolase did not catalyze the conversion of tyrosine to DOPA and does not appear to have a role in DOPA synthesis.     

DOPA synthesis in non-producer cultures of Mucuna deeringiana

Cell cultures of Mucuna deeringiana on various media fail to accumulate DOPA or stizolobic acid. Radiotracer studies prove that both compounds are being synthesized in the cells; however, the apparent lack of production is due to metabolic emphasis on catabolism instead of storage.     

Effect of p-aminophenols on tyrosinase activity

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure–activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver–Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.     

Monophenolase activity of avocado polyphenol oxidase

Polyphenol oxidase of avocado mesocarp catalyses (a) the orthohydroxylation of monophenols like l-tyrosine, d-tyrosine, tyramine and p-cresol, and (b) the oxidation of the corresponding o-dihydroxyphenols to quinones. The rate of step b is much greater than that of step a. The hydroxylation of monophenols occurs after a lag period. DOPA or ascorbate effectively eliminate the lag but not dl-6-methyltetrahydropteridine or tetrahydrofolic acid. At 1.66 × 10^?4 M, α,α-dipyridyl has no effect, while diethyldithiocarbamate at this concentration inhibits the hydroxylation reaction by 90%. The tyrosinase activity of avocado polyphenol oxidase is inactivated in the course of the reaction; this inactivation occurs faster and is more pronounced in the presence of exogenously added DOPA. This inactivation is partially prevented by a large excess of ascorbate. The K_m values indicate that tyramine, dopamine, p-cresol and 4-methyl catechol are better substrates for avocado polyphenol oxidase than tyrosine or DOPA.     

Conformational studies of sequential polypeptides containing l-β-3,4-dihydroxyphenyl-α-alanine (DOPA) and l-glutamic acid

The conformations of random and sequential copolypeptides containing l-β-3,4-dihydroxyphenyl-α-alanine (DOPA) and l-glutamic acid (Glu) as well as their protected precursors have been investigated mainly by means of circular dichroism (c.d.) spectroscopy in chloroform or trimethylphosphate as solvent. Protected and deprotected DOPA-containing polypeptides showed a positive ellipticity hand at 285 nm due to the stacked side-chains. The c.d. behaviour depended on the amino acid sequence as well as the amino acid composition. In random copolypeptides, ellipticities versus DOPA content showed a smooth variation, without any sharp changes. This supported the conclusion that poly(DOPA) is a right-handed helix. Although the ellipticities were low compared with the values of a typical α-helix, deprotected DOPA-containing sequential polypeptides are helical from the results of the induced dichroic band at 285 nm and the infrared amide I and II absorption bands. The results obtained were compared with those of sequential polypeptides containing l-tyrosine and l-Glu.     

Biosynthesis and Genetic Incorporation of 3,4-Dihydroxy-L-Phenylalanine into Proteins in Escherichia coli

While 20 canonical amino acids are used by most organisms for protein synthesis, the creation of cells that can use noncanonical amino acids (ncAAs) as additional protein building blocks holds great promise for preparing novel medicines and for studying complex questions in biological systems. However, only a small number of biosynthetic pathways for ncAAs have been reported to date, greatly restricting our ability to generate cells with ncAA building blocks. In this study, we report the creation of a completely autonomous bacterium that utilizes 3,4-dihydroxy-L-phenylalanine (DOPA) as its 21st amino acid building block. Like canonical amino acids, DOPA can be biosynthesized without exogenous addition and can be genetically incorporated into proteins in a site-specific manner. Equally important, the protein production yields of DOPA-containing proteins from these autonomous cells are greater than those from cells exogenously fed with 9 mM DOPA. The unique catechol moiety of DOPA can be used as a versatile handle for site-specific protein functionalizations via either oxidative coupling or strain-promoted oxidation-controlled cyclooctyne-1,2-quinone (SPOCQ) cycloaddition reactions. We further demonstrate the use of these autonomous cells in preparing fluorophore-labeled anti-human epidermal growth factor 2 (HER2) antibodies for the detection of HER2 expression on cancer cells.     

Effect of light and exogenously applied precursors on amaranthin synthesis in Amaranthus caudatus

Light stimulates the synthesis of amaranthin in Amaranthus caudatus var. viridis. Evidence suggests that this stimulation is markedly dependent on seedling age. Synthesis is controlled by both a “low-energy” red/far-red reversible phytochrome system and an HER at least partially under phytochrome control. In seedlings exposed to light, synthesis is promoted by exogenously applied DOPA and tyrosine. It is suggested that at least two light-promoted reactions occur in the biosynthetic pathway; one between tyrosine and DOPA and a second between DOPA and amaranthin.     

Phenylalanine Metabolism Regulates Reproduction and Parasite Melanization in the Malaria Mosquito

The blood meal of the female malaria mosquito is a pre-requisite to egg production and also represents the transmission route for the malaria parasite. The proper and rapid assimilation of proteins and nutrients in the blood meal creates a significant metabolic challenge for the mosquito. To better understand this process we generated a global profile of metabolite changes in response to blood meal of Anopheles gambiae, using Gas Chromatography-Mass Spectrometry (GC-MS). To disrupt a key pathway of amino acid metabolism we silenced the gene phenylalanine hydroxylase (PAH) involved in the conversion of the amino acid phenylalanine into tyrosine. We observed increased levels of phenylalanine and the potentially toxic metabolites phenylpyruvate and phenyllactate as well as a reduction in the amount of tyrosine available for melanin synthesis. This in turn resulted in a significant impairment of the melanotic encapsulation response against the rodent malaria parasite Plasmodium berghei. Furthermore silencing of PAH resulted in a significant impairment of mosquito fertility associated with reduction of laid eggs, retarded vitellogenesis and impaired melanisation of the chorion. Carbidopa, an inhibitor of the downstream enzyme DOPA decarboxylase that coverts DOPA into dopamine, produced similar effects on egg melanization and hatching rate suggesting that egg chorion maturation is mainly regulated via dopamine. This study sheds new light on the role of amino acid metabolism in regulating reproduction and immunity.     

A Simple Radioenzymatic Method to Measure Picogram Amounts of DOPA in Brain and Biological Fluids

A simple radioenzymatic method for the determination of DOPA is described. The method is based on the conversion of DOPA to 3-O-[methyl-³H]DOPA by catechol-O-methyltransferase in the presence of S-adenosyl-[methyl-³H]methionine and purification of the labelled product by Sephadex G10 and Dowex 50 W × 4 ion exchange resin. The method has been applied to the assay of endogenous DOPA in different brain areas and to measuring DOPA accumulation after inhibition of aromatic amino acid DOPA decarboxylase.     

DOPA and tyrosine decarboxylase activity in tissues of Periplanet a americana in relation to cuticle formation and ecdysis

DOPA decarboxylase activity in haemolymph and integument was low in last instar and early pharate adult Periplaneta americana, but began to increase shortly before ecdysis. Decarboxylation rates of l-DOPA, about 10 times the larval level by the start of ecdysis, reached a peak about 6 hr afterward, coinciding with the main period of cuticular sclerotization. Activity decreased rapidly during the next 18 hr, then decreased gradually for several days. Haemolymph DOPA decarboxylase activity was about four times greater than the integument, based on tissue dry weights. The fat body and gut tissues had low DOPA decarboxylase activity in all ages tested, and this did not increase at ecdysis. Tyrosine decarboxylase activity was significant only in the haemolymph and at consistently low levels.DOPA decarboxylase, therefore, apparently plays a major rôle in production of catecholamine derivatives for cuticular sclerotization in P. americana, while tyrosine decarboxylation is minor. Both haemolymph and integument appear to be important sites of dopamine biosynthesis.     

Presence of 3,4-dihydroxyphenylalanine and 3,4,5-trihydroxyphenylalanine in a coelenterate nervous system: Possible tyrosinase-mediated accumulation

3,4-dihydroxyphenylalanine (DOPA), 3,4,5-trihydroxyphenylalanine (5-OH-DOPA), 5-S-cysteinylDOPA (5-SC.D) and 2-S-cysteinylDOPA (2-SC.D) in the tentacles of the sea anemone, Metridium senile, were studied by the combined use of differential centrifugation of tissue homogenates, ultracentrifugation on sucrose density gradients, HPLC and electron microscopy. DOPA, 5-OH-DOPA and o-diphenol:O₂ oxidoreductase (Tyrosinase) were enriched in fractions containing membranes and subcellular particles, and the cysteinylDOPAs in the cytosol fractions. Ultrastructurally studied fractions rich in DOPA and 5-OH-DOPA contained large numbers of highly osmium-reducing vesicles. Identical structures were localized in ectodermal nerves and epidermal sensory cells.The results suggest that previous findings of catecholderivatives in the tentacles of Metridium and other sea anemone species by histochemical methods, are explained by a tyrosinase-based accumulation of DOPA and 5-OH-DOPA in the ectodermal nerve net. These substances are confined in specific compartments (vesicles) in the neurons and sensory cells.     

Some enzymes of general metabolism in the latex of Papaver somniferum

Enzymes of general metabolism have been determined in the latex of Papaver somniferum in an attempt to elucidate further the nature of the 1000 g130 min organelles and their role in alkaloid biogenesis. A number of enzymes involved in the glyoxylic acid and tricarboxylic acid cycles have been found, namely, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, malate dehydrogenase and isocitrate lyase. Two enzymes of glycolysis, namely, pyruvate kinase and lactate dehydrogenase, as well as enzymes associated with peroxisomes (glyoxylate reductase, catalase) and lysosomes (arylesterase, acid phosphatase) have been studied. Finally, some enzymes previously reported as occurring in poppy seedlings have been investigated, namely peroxidase, glutamate—oxaloacetate and glutamate-pyruvate transaminases, together with phenylalanine, tyrosine, DOPA and glutamic acid decarboxylases.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Changed betaxanthin pattern in violet flowers of Portulaca grandiflora after the feeding of DOPA

The administration of DOPA to violet flowers of Portulaca grandiflora led to the biosynthesis of betaxanthins not present in untreated plant material. Among them vulgaxanthin II but no dopaxanthin could be identified. DOPA serves as a precursor of the dihydropyridine moiety of the new betaxanthins. Its role as an elicitor of betaxanthin biosynthesis is discussed.     

alpha,beta-Dehydro-3,4-dihydroxyphenylalanine derivatives: potential schlerotization intermediates in natural composite materials.

Proteins containing the post-translationally modified amino acid L-3,4-dihydroxyphenylalanine (DOPA) undergo autosclerotization as a means of assuring cohesive resilience in many structural matrices found in nature. To explore the chemical mechanism of sclerotization, we examined the oxidation products of relatively simple analogs of a peptidyl DOPA residue, such as N-acetylDOPA ethyl ester and N-acetyldopamide, together with those of several oligopeptides. Oxidation, induced by either of two catecholoxidases or by sodium periodate, resulted in the Lewis base catalyzed formation of derivatives of the unusual amino acid 3,4-dihydroxy-alpha,beta-dehydroDOPA (delta DOPA). The N-acetyl delta DOPA ethyl ester representative of this group of derivatives was characterized by NMR and uv spectroscopy. A variety of peptides developed analogous uv spectra upon oxidation. A similar reaction was observed upon oxidation of 3,4-dihydroxyphenylpropanoic (dihydrocaffeic) acid, but not after oxidation of N-acetyldopamine. Evidence is presented that this conversion is the result of a rearrangement of the DOPA quinone moiety to its delta DOPA tautomer, and that this tautomerization can be a dominant fate for peptidyl DOPA quinone, provided a Lewis base catalyst is available and competing reactions are minimized. Formation of delta DOPA in natural or synthetic polymers would increase the variety of crosslinks available to sclerotizing matrices. delta DOPA has been found in naturally occurring oligopeptides isolated by other workers from several marine species.     

Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H₂O as electron donor for the photoreduction of NADP, (b) NADP replaced O₂ as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O₂ at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.     

Transmitter-Like Basal and K+-Evoked Release of 3,4-Dihydroxyphenylalanine from the Striatum in Conscious Rats Studied by Microdialysis

Using microdialysis and HPLC, characteristics of the release of endogenous 3,4-dihydroxyphenylalanine (DOPA) from striatum in conscious rats were studied in comparison with those of 3,4-dihydroxyphenylethylamine (dopamine; DA). Purified L-aromatic amino acid decarboxylase (AADC) converted a putative peak of DOPA to DA. The retention time of DOPA differed from that of DA and major metabolites of DA and norepinephrine. The DOPA peak of dialysates comigrated with that of authentic DOPA when the pH of the HPLC buffer was modified. The ratio of the basal release of DOPA:DA was 1:2. 3-Hydroxybenzylhydrazine (NSD-1015; 100 mg/kg, i.p.), an AADC inhibitor, markedly increased the basal release of DOPA but produced no effect on DA. The basal release of DOPA was markedly decreased by alpha-methyl-p-tyrosine (200 mg/kg, i.p.), substantially tetrodotoxin (1 microM) sensitive, and Ca2+ (removal plus 12.5 mM Mg2+ addition) dependent. Fifty millimolar K+ released DOPA and this release was also Ca2+ dependent. These characteristics of the basal and evoked release of DOPA were similar to those of DA. The ratio of the evoked release of DOPA:DA was 1:3. These results indicate that DOPA is released under physiological conditions and by K(+)-induced depolarization in a manner similar to that for transmitter DA from striatum in freely moving rats.     

Microdialysis Monitoring of 3,4-Dihydroxyphenylalanine Accumulation After Decarboxylase Inhibition: A Means to Estimate In Vivo Changes in Tyrosine Hydroxylase Activity of the Rat Locus Ceruleus

Abstract: An on-line microdialysis approach was developed to estimate changes in tyrosine hydroxylase activity in the locus ceruleus noradrenergic neurons of anesthetized rats by measuring the 3,4-dihydroxyphenylalanine (DOPA) acumulation in the extracellular fluid during perfusion of an aromatic amino acid decarboxylase inhibitor through a dialysis probe. The aromatic amino acid decarboxylase inhibitor used was difluoromethyl-DOPA, which was shown to be more stable than NSD 1015 or Ro 4-4602 in the perfusion fluid. A 1-h perfusion of a 10⁻⁴ mol/L of difluoromethyl-DOPA solution induced a linear increase in DOPA concentration in the locus ceruleus dialysates that achieved a steady state within 1 h. The identity of DOPA accumulated in dialysates during aromatic amino acid decarboxylase inhibition was confirmed by the disappearance of the chromatographic peak when DOPA formation was blocked by the administration of α-methyl- p -tyrosine. Systemic administration of the α₂-antagonist piperoxane before difluoromethyl-DOPA perfusion markedly increased the DOPA concentration during both the accumulation and the steady-state periods, showing that the present technique is a suitable in vivo approach to monitor changes in tyrosine hydroxylase activity occurring in the locus ceruleus neurons.     

Effects of dithiothreitol on dopa oxidase activity from potato tubers

A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10^?2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all.