Regulation of mammalian melanogenesis. I: Partial purification and characterization of a dopachrome converting factor: dopachrome tautomerase

A protein that catalyzes the decoloration of dopachrome has been partially purified from B16 mouse melanoma tumors. The enzyme is preferentially associated to the melanosomes, but it is also found in the microsomal and cytosolic fractions of cellular homogenates. The protein is clearly different from tyrosinase, and should be related to the dopachrome oxidoreductase (Barber et al. (1984) J. Invest. Dermatol. 83, 145-149) and the dopachrome conversion factor (Korner and Pawelek (1980) J. Invest. Dermatol. 75, 192-195) since the reaction product of dopachrome conversion is 5,6-dihydroxyindole-2-carboxylic acid. The protein appears to have an oligomeric structure, with a molecular mass slightly higher than 300 kDa estimated by gel filtration, whereas the molecular mass of the monomer might be approx. 46 kDa estimated by SDS-PAGE electrophoresis. Its Km for dopachrome is around 100 microM. The enzyme is competitively inhibited by indoles and is unaffected by metal chelators. It also has the ability to increase the amount of melanin formed from L-tyrosine by melanoma tyrosinase, and therefore, cannot be considered an 'indole blocking factor' as was suggested for the related dopachrome oxidoreductase. Since the reaction catalyzed by the enzyme is a tautomeric shift on dopachrome, we would propose dopachrome tautomerase (EC 5.3.2.3) as the most precise and informative name.     

A spectrophotometric assay for mammalian tyrosinase utilizing the formation of melanochrome from L-dopa

A simple spectrophotometric method for a rapid determination of tyrosinase (EC 1.14.18.1) is described. The basis of the assay is the incubation of the enzyme with L-dopa in the presence of an optimal concentration of Zn2+ ions and the measurement of the formation of melanochrome, as indicated by the rise in absorbance at 540 nm. Final absorbance change reflects probably two activities of tyrosinase: the oxidation of dopa to dopaquinone and the conversion of 5,6-dihydroxyindole to melanochrome. Using a purified preparation from hamster melanoma, the assay was found to be more sensitive than the commonly used dopachrome assay. Comparison with some other currently available methods for assaying tyrosinase is presented and potential applications of the assay are discussed.     

The role of pH in the melanin biosynthesis pathway

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.     

A Method for Detecting Dopaminechrome Isomerase Activity on Gels

Melanogenesis involves oxidation of 3,4-dihydroxyphenylalanine (dopa) to dopachrome which then is converted into 5,6-dihydroxyindole by dopachrome isomerase. 5,6-Dihydroxyindole is oxidized to its quinone which in turn is metabolized nonenzymatically to melanin. In addition to dopachrome isomerase, a new dopminechrome isomerase activity involved in the conversion of dopaminechrome into 5,6-dihydroxyindole has been observed in the larva of Rhinoceros oryctes. This dopaminechrome isomerase differs from dopachrome isomerase in its electrophoretic mobility and substrate specificity. The present study reports a specific, sensitive and rapid staining method for detecting dopaminechrome isomerase activity after electrophoresis. Using this new method, the presence of the dopaminechrome isomerase activity, which is involved in melanogenesis, could easily be detected by staining tyrosinase embedded native gels in dopamine solution. Tyrosinase entrapped in the gels converts dopamine in dopaminechrome. The dopaminechrome isomerase separated in the gels catalyzes dopaminechrome to 5,6-dihydroxyindole which is oxidized further by tyrosinase to colored melanochrome. The dopaminechrome isomerase appears as a bluish purple band against a pink background.     

After dopachrome?

Dopachrome, an intermediate in melanin biosynthesis, exhibits some unusual properties. At physiologic pH (e.g., pH 6-8) it is unstable and spontaneously loses its carboxyl group to form 5,6-dihydroxyindole (DHI) and CO2. However, over this same pH range, if various metals or a melanocyte-specific enzyme are present, it rapidly rearranges to its isomer form--5,6-dihydroxyindole-2-carboxylic acid (DHICA)--which is far more stable than dopachrome in its ability to retain the carboxyl group. Whether or not the carboxyl group is retained could have important implications for the regulation of melanogenesis, since in the presence of oxygen DHI spontaneously forms a black precipitate, whereas DHICA forms a golden-brown solution. The solubility of "DHICA-melanin" is due to the presence of carboxyl groups, which provide negative charges and hydrophilicity. Thus, in vivo, the extent to which dopachrome is converted to DHI or DHICA may well influence the solubility and color of the melanin formed. The purpose of this article is to review recent findings in these areas and to discuss the possible significance of dopachrome conversion in the regulation of melanogenesis and color formation.     

Electrochemical identification of dopachrome isomerase in Drosophila melanogaster

A principal reaction in the eumelanin biosynthetic pathway is the conversion of dopachrome (DC) to dihydroxyindole(s). Dopachrome isomerase (DI), the enzyme that catalyzes this reaction, was detected for the first time in larvae of D. melanogaster. Unlike the enzyme from B16 mouse melanoma cells which converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), the insect enzyme forms 5,6-dihydroxyindole (DHI). The activity of the insect DI was linear through 15 min incubation, and the amount of DHI produced was proportional to the amount of enzyme that was incorporated into the reaction mixtures.     

Function of dopachrome oxidoreductase and metal ions in dopachrome conversion in the eumelanin pathway

The conversion of dopachrome (DC) in the eumelanin pathway has been analyzed to determine the specific product and the role of enzyme control. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) were quantitated by HPLC with fluorescent detection, after DC incubation with heated and unheated preparations of B-16 melanoma derived dopachrome oxidoreductase (DCOR). The enzyme-catalyzed reaction produced DHICA as the major product, while DHI formed with the spontaneous reaction. It had originally been suggested that the major product of DC conversion was DHI, with DHICA being formed as a minor product of this conversion [Raper, H.S. (1927) Biochem. J. 21, 89-96]. Copper, nickel, and cobalt ions promoted conversion of DC, with nickel simulating DCOR activity. Removal of free ions from unheated DCOR did not alter DC conversion. We conclude that the major product of DC conversion is DHICA and that DCOR is responsible for this conversion.     

The Mouse Brown (b) Locus Protein Functions as a Dopachrome Tautomerase

The mouse b locus controls black/brown coat coloration. Its product, the b-protein or TRP-1, has significant homology to tyrosinase, and this has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b-protein. We were unable to con-firm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.     

Mechanism of dopachrome tautomerization into 5,6-dihydroxyindole-2-carboxylic acid catalyzed by Cu(II) based on quantum chemical calculations

Background

Tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) is a biologically crucial reaction relevant to melanin synthesis, cellular antioxidation, and cross-talk among epidermal cells. Since dopachrome spontaneously converts into 5,6-dihydroxyindole (DHI) via decarboxylation without any enzymes at physiologically usual pH, the mechanism of how tautomerization to DHICA occurs in physiological system is a subject of intense debate. A previous work has found that Cu(II) is an important factor to catalyze the tautomerization of dopachrome to DHICA. However, the effect of Cu(II) on the tautomerization has not been clarified at the atomic level.

Methods

We propose the reaction mechanism of the tautomerization to DHICA by Cu(II) from density functional theory-based calculation.

Results

We clarified that the activation barriers of α-deprotonation, β-deprotonation, and decarboxylation from dopachrome are significantly reduced by coordination of Cu(II) to quinonoid oxygens (5,6-oxygens) of dopachrome, with the lowest activation barrier of β-deprotonation among them. In contrast to our previous work, in which β-deprotonation and quinonoid protonation (O5/O6-protonation) were shown to be important to form DHI, our results show that the Cu(II) coordination to quinonoid oxygens inhibits the quinonoid protonation, leading to the preference of proton rearrangement from β-carbon to carboxylate group but not to the quinonoid oxygens.

Conclusion

Integrating these results, we conclude that dopachrome tautomerization first proceeds via proton rearrangement from β-carbon to carboxylate group and subsequently undergoes α-deprotonation to form DHICA.

General significance

This study would provide the biochemical basis of DHICA metabolism and the generalized view of dopachrome conversion which is important to understand melanogenesis.     

Synthesis, discovery and mechanism of 2,6-dimethoxy-N-(4-methoxyphenyl)benzamide as potent depigmenting agent in the skin

In this study, a new skin-depigmenting agent, 2,6-dimethoxy-N-(4-methoxyphenyl)benzamide (DMPB), was synthesized using a combination of benzoic acid and aniline. DMPB exhibited significant depigmentation ability on the UV B-induced hyperpigmentation of the brown guinea pig skin. In addition, the 100ppm treatment with this compound had a 30% inhibitory effect on melanin pigment generation in the melan-a cell line without significant cell toxicity. To search for relationship with the depigmentation, the effects of DMPB on the tyrosinase and dopachrome tautomerase were evaluated. DMPB had no effect on tyrosinase. However, it accelerated dopachrome transformation into 5,6-dihydroxyindole-2-carboxylic acid (DHICA) in the presence of dopachrome tautormerase. In addition, intracellular level of dopachrome tautomerase in melan-a cells was increased by treatment of DMPB. These results suggest that the pigment-lightening effects of DMPB might be due to biased production of DHICA-eumelanin induced by dopachrome tautormerase activation.     

蛋白质二硫键异构酶的纯化和活力测定

 从500g新鲜牛肝制得蛋白质二硫键异构酶(PDI,EC 5.3.4.1)98mg。该酶制剂在SDS-聚丙烯酰胺凝胶电泳中表现为亚基分子量62,000的均一条带。在260nm追踪,因二硫键错接而失活的牛胰核糖核酸酶A,经PDI作用使其二硫键重排恢复活力,从而催化酵母RNA的水解来测定PDI活力。这种单波长法比文献中介绍的追踪A_(260)—A_(280)的双波长法更为灵敏方便。酶的克分子消光系数ε_M=1.03×10~5(pH7.5),其比活性为1400单位/克蛋白质。     

Quinone methide as a new intermediate in eumelanin biosynthesis

The conversion of dopachrome to dihydroxyindole(s), a key reaction in eumelanin biosynthetic pathway, has been shown to be under the control of dopachrome conversion factor. Dopachrome conversion factor isolated from the hemolymph of Manduca sexta larvae, which is devoid of any tyrosinase activity, exhibits a narrow substrate specificity and readily bleaches the iminochromes derived from the oxidation of L-dopa, L-dopa methyl ester, and alpha-methyl-L-dopa, but failed to attack the corresponding D-isomers. The product formed in the case of L-dopachrome was identified to be 5,6-dihydroxyindole. Therefore, aromatization of dopachrome seems to accompany its decarboxylation as well. However, the enzyme also converts L-dopachrome methyl ester to an indole derivative indicating that it can deprotonate the alpha-hydrogen when the carboxyl group is blocked. These results are accounted for by the transient formation and further transformation of a reactive quinone methide intermediate during the dopachrome conversion factor-catalyzed reaction. The fact that the enzyme-catalyzed conversion of alpha-methyl dopachrome methyl ester (where both decarboxylation and deprotonation are blocked) resulted in the generation of a stable quinone methide in the reaction mixture confirms this contention and supports our recent proposal that quinone methide and not indolenine is the key transient intermediate in the conversion of dopachrome to dihydroxyindole observed during melanogenesis.     

Synthesis and characterization of melanins from dihydroxyindole-2-carboxylic acid and dihydroxyindole.

Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 micron filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.).(ABSTRACT TRUNCATED AT 250 WORDS)     

Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

Comparative biochemistry of eumelanogenesis and the protective roles of phenoloxidase and melanin in insects

The phenolic biopolymer eumelanin is an important skin pigment found throughout the animal kingdom. The enzyme, tyrosinase, initiates melanogenesis in mammals. The biogenesis is assisted by a number of mammalian protein factors including dopachrome tautomerase and 5,6-dihydroxyindole-2-carboxylate oxidase. Invertebrates, such as insects, employ phenoloxidase and dopachrome (decarboxylating) isomerase for melanin biosynthesis. Recently generated molecular biological and biochemical data indicate that tyrosinase and phenoloxidase are distinctly different enzymes in spite of possessing both monophenol monooxygenase activity as well as o-diphenoloxidase activity. Similarly, insect dopachrome isomerase also differs significantly from its mammalian counterpart in several of its properties including the nature of the enzymatic reaction. In addition, there are considerable differences in the eumelanogenic pathways of these two animal groups that include the utility of substrates, use of dihydroxyindoles and the nature of eumelanin pigment. Thus, the biochemistry and molecular biology of melanogenesis in mammals and insects are significantly different. The advantages of generating different eumelanin pigments and intermediates by the insects are discussed.     

The role of 2,4,5-trihydroxyphenylalanine in melanin biosynthesis.

Both 3,4-dihydroxyphenylalanine and 2,4,5-trihydroxyphenylalanine were oxidized with periodate and mushroom tyrosinase to determine whether the latter compound is an intermediate in melanin biosynthesis. Matrix analysis of the spectra obtained with a rapid scan spectrophotometer and comparison of the spectra of quinone intermediates with model quinones disclosed that, although 2,4,5-trihydroxyphenylalanine can be oxidized to 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome), this oxidation proceeds through a stable intermediate, 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone, which does not appear in the oxidation of 3,4-dihydroxyphenylalanine to dopachrome. Thus, these studies are in agreement with the original postulate, that 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone and leukodopachrome are the intermediates in the major pathway for dopachrome synthesis.     

Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.     

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.     

Rapid photometric assay of growth of Mycoplasma mycoides subsp. capri

A new spectrophotometric technique for evaluation of early growth in liquid culture of Mycoplasma mycoides subsp. capri has been developed. As turbidity does not appear until after incubation to 18 h the method utilizes the change in absorbance of the medium at 550 nm to monitor growth. The change in absorbance of the medium (which contains phenol red) occurs when the pH changes due to microbial growth. For measurement of growth at later stages when turbidity is proportional to number of colony forming units, two other wavelengths (450 nm and 700 nm) have been suggested.     

Rapid photometric assay of growth of Mycoplasma mycoides subsp. capri

A new spectrophotometric technique for evaluation of early growth in liquid culture of Mycoplasma mycoides subsp. capri has been developed. As turbidity does not appear until after incubation to 18 h the method utilizes the change in absorbance of the medium at 550 nm to monitor growth. The change in absorbance of the medium (which contains phenol red) occurs when the pH changes due to microbial growth. For measurement of growth at later stages when turbidity is proportional to number of colony forming units, two other wavelengths (450 nm and 700 nm) have been suggested.