A three‐dimensional model of mammalian tyrosinase active site accounting for loss of function mutations

Tyrosinases are the first and rate‐limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non‐mammalian tyrosinases provides an active site model accounting for loss‐of‐function mutations. According to the model, the copper‐binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH‐groups of the imidazole rings of the copper‐binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution ³⁷⁴MS³⁷⁵→³⁷⁴GG³⁷⁵ or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.     

Bacterial tyrosinases

Tyrosinases are nearly ubiquitously distributed in all domains of life. They are essential for pigmentation and are important factors in wound healing and primary immune response. Their active site is characterized by a pair of antiferromagnetically coupled copper ions, CuA and CuB, which are coordinated by six histidine residues. Such a "type 3 copper centre" is the common feature of tyrosinases, catecholoxidases and haemocycanins. It is also one of several other copper types found in the multi-copper oxidases (ascorbate oxidase, laccase). The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity). The best-known function is the formation of melanins from L-tyrosine via L-dihydroxyphenylalanine (L-dopa). The complicated hydroxylation mechanism at the active centre is still not completely understood, because nothing is known about their tertiary structure. One main reason for this deficit is that hitherto tyrosinases from eukaryotic sources could not be isolated in sufficient quantities and purities for detailed structural studies. This is not the case for prokaryotic tyrosinases from different Streptomyces species, having been intensively characterized genetically and spectroscopically for decades. The Streptomyces tyrosinases are non-modified monomeric proteins with a low molecular mass of ca. 30kDa. They are secreted to the surrounding medium, where they are involved in extracellular melanin production. In the species Streptomyces, the tyrosinase gene is part of the melC operon. Next to the tyrosinase gene (melC2), this operon contains an additional ORF called melC1, which is essential for the correct expression of the enzyme. This review summarizes the present knowledge of bacterial tyrosinases, which are promising models in order to get more insights in structure, enzymatic reactions and functions of "type 3 copper" proteins in general.     

Crystal structure of Agaricus bisporus mushroom tyrosinase: identity of the tetramer subunits and interaction with tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 ? resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ～392 residues and two L subunits of ～150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ～100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 ? away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.     

Structure–activity relationships of the thujaplicins for inhibition of human tyrosinase

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, β, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC₅₀ of 1.15 μM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC₅₀ = 571.17 μM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.     

Identification of active site residues involved in metal cofactor binding and stereospecific substrate recognition in Mammalian tyrosinase. Implications to the catalytic cycle.

Tyrosinase (Tyr) and tyrosinase-related proteins (Tyrps) 1 and 2 are the enzymes responsible for mammalian melanogenesis. They display high similarity but different substrate and reaction specificities. Loss-of-function mutations lead to several forms of albinism or other pigmentation disorders. They share two conserved metal binding sites (CuA and CuB) which, in Tyr, bind copper. To define some structural determinants for these differences, we mutated Tyr at selected residues on the basis of (i) conservation of the original residues in most tyrosinases, (ii) their nonconservative substitution in the Tyrps, and (iii) their possible involvement as an endogenous bridge between the copper pair. Two mutations at the CuA site, S192A and E193Q, did not affect Tyr activities, thus excluding S192 and E193 as endogenous ligands of the copper pair. Concerning CuB, the H390Q mutation completely abolished Tyr activity, whereas Q378H and H389L mutants showed 10-20% residual specific activities. Their kinetic behavior suggests that (i) H390 is the actual third ligand for CuB, (ii) H389 is critical for stereospecific recognition of o-diphenols but not monophenols, and (iii) the involvement in metal binding of the central extra H residue at the Tyrps CuB site is unlikely. However, replacement of Q (in Tyr) by H (in Tyrps) greatly diminished the affinity for L-dopa, consistent with the low/null tyrosinase activity of the Tyrps. These are the first data showing a physical difference in docking of mono- and o-diphenols to the Tyr active site, and they are used to propose a revised scheme of the catalytic cycle.     

Homology models of four Agaricus bisporus tyrosinases

Partially purified tyrosinase from the white button mushroom Agaricus bisporus is available commercially and is a widely used experimental model for the study of tyrosinase. The structure of an H₂L₂ tetrameric form of the mushroom enzyme was recently determined by X-ray crystallography. In this structure the two H subunits originate from the PPO3 gene, and the two L subunits are formed by a protein of unknown function with a lectin-like fold. However, the X-ray structures and oligomeric states of the mushroom PPO1, PPO2, PPO4, and PPO5 gene products remain unknown. Commercial mushroom tyrosinase powder is a mixture containing several or all of these tyrosinases, so knowledge of their structures should provide insight regarding interpretation of experimental data generated using commercial preparations of the enzyme. The PPO3 structure (H-subunit) was used as a template to generate homology models for the structures of the other four tyrosinases, and the resulting structural models were evaluated. Due to the moderate to high percentage of sequence identity (∼37-76%) between PPO3 and the other four tyrosinases, the backbone conformations of the predicted structures are very similar to that of PPO3. The alpha carbons of the six copper-coordinating histidines in the active site are positioned properly in the predicted structures, but their side chains are not oriented optimally for copper binding in some cases. Thus, the models are likely to provide an accurate representation of the actual tertiary structures, but they may have limited use in studies involving docking of substrates or inhibitors in the active site. Comparison of the homology models to the structure of molluscan hemocyanin enabled a prediction of the orientation of the enzyme's C-terminal domain over the active site in the latent enzyme.     

New insights into the active site structure and catalytic mechanism of tyrosinase and its related proteins

Tyrosinases are widely distributed in nature. They are copper‐containing oxidases belonging to the type 3 copper protein family, together with catechol oxidases and haemocyanins. Tyrosinases are essential enzymes in melanin biosynthesis and therefore responsible for pigmentation of skin and hair in mammals, where two more enzymes, the tyrosinase‐related proteins (Tyrps), participate in the pathway. The structure and catalytic mechanism of mammalian tyrosinases have been extensively studied but they are not completely understood because of the lack of information on the tertiary structure. The availability of crystallographic data of one plant catechol oxidase and one bacterial tyrosinase has improved the model of the three‐dimensional structure of the active site of the enzyme. Furthermore, sequence comparison of tyrosinase and the Tyrps reveals that the three orthologue proteins share many key structural features, because of their common origin from an ancestral gene, although the specific residues responsible for their different catalytic capabilities have not been identified yet. This review summarizes our current knowledge of tyrosinase and Tyrps structure and function and describes the catalytic mechanism of tyrosinase and Dct/Tyrp2, which are better characterized.     

A tyrosinase with an abnormally high tyrosine hydroxylase/dopa oxidase ratio

The sequencing of the genome of Ralstonia solanacearum[Salanoubat M, Genin S, Artiguenave F, et al. (2002) Nature 415, 497-502] revealed several genes that putatively code for polyphenol oxidases (PPOs). This soil-borne pathogenic bacterium withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions at the active site. Generation of null mutants in those genes by homologous recombination mutagenesis and protein purification allowed us to correlate each gene with its enzymatic activity. In contrast with all tyrosinases so far studied, the enzyme NP_518458 shows higher monophenolase than o-diphenolase activity and its initial activity does not depend on the presence of l-dopa cofactor. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual monophenolase activity, behaving as a catechol oxidase. These catalytic characteristics are discussed in relation to two other characteristics apart from the six conserved histidines. One is the putative presence of a seventh histidine which interacts with the carboxy group on the substrate and controls the preference for carboxylated and decarboxylated substrates. The second is the size of the residue isosteric with the aromatic F261 reported in sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.     

Cloning and Molecular Characterization of a SDS‐Activated Tyrosinase from Marinomonas mediterranea

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper‐binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS‐dependent conformational change that facilitates the substrate accessibility to the dicopper active site.     

Cloning and molecular characterization of a SDS-activated tyrosinase from Marinomonas mediterranea

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper-binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS-dependent conformational change that facilitates the substrate accessibility to the dicopper active site.     

The crystal structure of an extracellular catechol oxidase from the ascomycete fungus Aspergillus oryzae

Catechol oxidases (EC 1.10.3.1) catalyse the oxidation of o-diphenols to their corresponding o-quinones. These oxidases contain two copper ions (CuA and CuB) within the so-called coupled type 3 copper site as found in tyrosinases (EC 1.14.18.1) and haemocyanins. The crystal structures of a limited number of bacterial and fungal tyrosinases and plant catechol oxidases have been solved. In this study, we present the first crystal structure of a fungal catechol oxidase from Aspergillus oryzae (AoCO4) at 2.5-Å resolution. AoCO4 belongs to the newly discovered family of short-tyrosinases, which are distinct from other tyrosinases and catechol oxidases because of their lack of the conserved C-terminal domain and differences in the histidine pattern for CuA. The sequence identity of AoCO4 with other structurally known enzymes is low (less than 30 %), and the crystal structure of AoCO4 diverges from that of enzymes belonging to the conventional tyrosinase family in several ways, particularly around the central α-helical core region. A diatomic oxygen moiety was identified as a bridging molecule between the two copper ions CuA and CuB separated by a distance of 4.2–4.3 Å. The UV/vis absorption spectrum of AoCO4 exhibits a distinct maximum of absorbance at 350 nm, which has been reported to be typical of the oxy form of type 3 copper enzymes.     

Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fül?p, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fül?p, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fül?p, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.     

The existence of apotyrosinase in the cytosol of Harding-Passey mouse melanoma melanocytes and characteristics of enzyme reconstitution by Cu(II)

This paper reports the effect of Cu(II) supplementation on the tyrosinase isozymes from Harding-Passey mouse melanoma. The dopa-oxidase activity of the microsomal and soluble isozymes is increased by incubation with Cu(II), whereas the activity of the unique 'in vivo' melanin-forming isozyme, bound to melanosomes, is not. Other divalent cations are ineffective in increasing the dopa-oxidase activity of tyrosinases. These results indicate the existence of a mixture of tyrosinase and apotyrosinase in the cytosol of melanocytes before reaching the melanosome. The paucity of Cu(II) in the cytosol could be one of the mechanisms of regulation contributing to avoid the formation of melanin outside the melanosome. Some kinetic characteristics of the enzymatic reconstitution of soluble and microsomal isozymes by Cu(II) are also studied, and the results suggest that the glycosylation of apotyrosinase during its maturation yields a conformational change favouring the binding of Cu(II) at the enzyme active site, by lowering the activation energy of the reconstitution reaction.     

Molecular structure of the rat vitamin D receptor ligand binding domain complexed with 2-carbon-substituted vitamin D3 hormone analogues and a LXXLL-containing coactivator peptide

We have determined the crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor in ternary complexes with a synthetic LXXLL-containing peptide and the following four ligands: 1alpha,25-dihydroxyvitamin D(3); 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D(3) (2MD); 1alpha-hydroxy-2-methylene-19-nor-(20S)-bishomopregnacalciferol (2MbisP), and 2alpha-methyl-19-nor-1alpha,25-dihydroxyvitamin D(3) (2AM20R). The conformation of the LBD is identical in each complex. Binding of the 2-carbon-modified analogues does not change the positions of the amino acids in the ligand binding site and has no effect on the interactions in the coactivator binding pocket. The CD ring of the superpotent analogue, 2MD, is tilted within the binding site relative to the other ligands in this study and to (20S)-1alpha,25-dihydroxyvitamin D(3) [Tocchini-Valentini et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 5491-5496]. The aliphatic side chain of 2MD follows a different path within the binding site; nevertheless, the 25-hydroxyl group at the end of the chain occupies the same position as that of the natural ligand, and the hydrogen bonds with histidines 301 and 393 are maintained. 2MbisP binds to the receptor despite the absence of the 25-hydroxyl group. A water molecule is observed between His 301 and His 393 in this structure, and it preserves the orientation of the histidines in the binding site. Although the alpha-chair conformer is highly favored in solution for the A ring of 2AM20R, the crystal structures demonstrate that this ring assumes the beta-chair conformation in all cases, and the 1alpha-hydroxyl group is equatorial. The peptide folds as a helix and is anchored through hydrogen bonds to a surface groove formed by helices 3, 4, and 12. Electrostatic and hydrophobic interactions between the peptide and the LBD stabilize the active receptor conformation. This stablization appears necessary for crystal growth.     

Tyrosinase/catecholoxidase activity of hemocyanins: structural basis and molecular mechanism

The enzymes tyrosinase, catecholoxidase and hemocyanin all share similar active sites, although their physiological functions differ. Hemocyanins serve as oxygen carrier proteins, and tyrosinases and catecholoxidases (commonly referred to as phenoloxidases in arthropods) catalyze the hydroxylation of monophenols or the oxidation of o-diphenols to o-quinones, or both. Tyrosinases are activated in vivo by limited proteolytic cleavage, which might open up substrate access to the catalytic site. It has recently been demonstrated that if hemocyanins are subjected to similar proteolytic treatments (in vitro) they also exhibit at least catecholoxidase reactivity. On the basis of their molecular structures, hemocyanins are used as model systems to understand the substrate-active-site interaction between catecholoxidases and tyrosinases.     

Catalytic properties of an organic solvent-resistant tyrosinase from Streptomyces sp. REN-21 and its high-level production in E. coli

An organic solvent-resistant tyrosinase (OSRT) from Streptomyces sp. REN-21 is a unique enzyme showing high activity in the presence of organic solvents. The OSRT-catalyzed oxidation of monophenols such as tyrosine-containing peptides and proteins was examined. The catalytic properties of OSRT were compared with those of mushroom tyrosinase. OSRT was shown to oxidize Gly-l-Tyr most effectively among four peptide substrates tested. On the other hand, mushroom tyrosinase showed the highest activity toward l-Tyr-Gly under the condition of 1 mM substrate. OSRT oxidized several proteins, including casein and hemoglobin, with relatively higher activity compared with mushroom tyrosinase under the condition of 1% (w/v) substrate. Thus, it was clarified that the catalytic properties of OSRT toward tyrosine-containing peptides and proteins are different from those of mushroom tyrosinase under these conditions. The OSRT-encoding gene operon was cloned, and found to consist of two genes, designated ORF-OSRT and ORF-393. The former encodes apo-OSRT, and the latter encodes the putative activator protein of apo-OSRT. A binuclear copper-binding site (type-3 copper site) characteristic of tyrosinases is contained in the deduced amino acid sequence for apo-OSRT. A high-level production system for the OSRT was constructed using pET20b(+) and Escherichia coli BL21(DE3)pLysS. Approximately 54 mg of active OSRT was synthesized in a 1-liter broth culture by this system. The properties of the recombinant OSRT were similar to those of the wild-type enzyme. In conclusion, we succeeded in constructing a high-level production system for OSRT.     

Functional analysis of the Burkholderia cenocepacia ZmpA metalloprotease

Burkholderia cenocepacia ZmpA is expressed as a preproenzyme typical of thermolysin-like proteases such as Pseudomonas aeruginosa LasB and Bacillus thermoproteolyticus thermolysin. The zmpA gene was expressed using the pPRO-EXHTa His(6) tag expression system, which incorporates a six-His tag at the N-terminal end of the protein, and recombinant ZmpA was purified using Ni-nitrilotriacetic acid affinity chromatography. Upon refolding of the recombinant His(6)-pre-pro-ZmpA (62 kDa), the fusion protein was autoproteolytically cleaved into 36-kDa (mature ZmpA) and 27-kDa peptides. Site-directed mutagenesis was employed to infer the identity of the active site residues of ZmpA and to confirm that the enzyme undergoes autoproteolytic cleavage. Oligonucleotide mutagenesis was used to replace H(465) with G(465) or A(465), E(377) with A(377) or D(377), or H(380) with P(380) or A(380). Mutagenesis of H(465), E(377), or H(380) resulted in the loss of both autocatalytic activity and proteolytic activity. ZmpA with either substitution in H(380) was not detectable in B. cenocepacia cell extracts. The activity of the recombinant ZmpA was inhibited by EDTA and 1,10 phenanthroline, indicating that it is a zinc metalloprotease. ZmpA, however, was not inhibited by phosphoramidon, a classical inhibitor of the thermolysin-like proteases. The refolded mature ZmpA enzyme was proteolytically active against various substrates including hide powder azure, type IV collagen, fibronectin, neutrophil alpha-1 proteinase inhibitor, alpha(2)-macroglobulin, and gamma interferon, suggesting that B. cenocepacia ZmpA may cause direct tissue damage to the host or damage to host tissues through a modulation of the host's immune system.     

Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus

An optical, ultrastructural, and biochemical study of the melanin accumulation nodules found in the kidney of the teleost fish Sparus auratus is presented. These nodules are randomly distributed in the interstitium of the renal tissue. They are formed by large aggregates of cells replete with melanin granules. The melanin granules occur singly or also in aggregates inside the cells. Most of the granules are electron-dense, but sometimes small electron-lucent spaces within them can be seen. Some secondary lysosomes and dendritic processes can also be observed. Biochemical studies have proved for the first time the existence of measurable tyrosinase activity in those nodules. That activity was assayed using three methods: tyrosine hydroxylation, dopa oxidation, and melanin formation. Furthermore, inhibitors of well-characterized plant and animal skin tyrosinases were effective agents for inhibiting those activities in fish kidney preparations. This finding supports the notion of the existence of true tyrosinase in the melanin accumulation nodules of this tissue. Taking into account the results obtained, the origin and functions of the melanin-containing cells found in the teleost fish kidney are discussed.     

Molecular docking studies of a phlorotannin, dieckol isolated from Ecklonia cava with tyrosinase inhibitory activity

In this study, the phlorotannin dieckol, which was isolated from the brown alga Ecklonia cava, was examined for its inhibitory effects on melanin synthesis. Tyrosinase inhibitors are important agents for cosmetic products. We therefore examined the inhibitory effects of dieckol on mushroom tyrosinase and melanin synthesis, and analyzed its binding modes using the crystal structure of Bacillus megaterium tyrosinase (PDB ID: 3NM8). Dieckol inhibited mushroom tyrosinase with an IC(50) of 20μM and was more effective as a cellular tyrosinase having melanin reducing activities than the commercial inhibitor, arbutin, in B16F10 melanoma cells, and without apparent cytotoxicity. It was found that dieckol behaved as a non-competitive inhibitor with l-tyrosine substrates. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and dieckol. These molecular modeling studies were successful (calculated binding energy value: -126.12kcal/mol), and indicated that dieckol interacts with His208, Met215, and Gly46. These results suggest that dieckol has great potential to be further developed as a pharmaceutical or cosmetic agent for use in dermatological disorders associated with melanin.