Copper-containing Monooxygenase Research Articles

Rokubacteria in Northern Peatlands: Habitat Preferences and Diversity Patterns.

Rokubacteria is a phylogenetic clade of as-yet-uncultivated prokaryotes, which are detected in diverse terrestrial habitats and are commonly addressed as members of the rare biosphere. This clade was originally described as a candidate phylum; however, based on the results of comparative genome analysis, was later defined as the order-level lineage, Rokubacteriales, within the phylum Methylomirabilota. The physiology and lifestyles of these bacteria are poorly understood. A dataset of 16S rRNA gene reads retrieved from four boreal raised bogs and six eutrophic fens was examined for the presence of the Rokubacteriales; the latter were detected exclusively in fens. Their relative abundance varied between 0.2 and 4% of all bacteria and was positively correlated with pH, total nitrogen content, and availability of Ca and Mg. To test an earlier published hypothesis regarding the presence of methanotrophic capabilities in Rokubacteria, peat samples were incubated with 10% methane for four weeks. No response to methane availability was detected for the Rokubacteriales, while clear a increase in relative abundance was observed for the conventional Methylococcales methanotrophs. The search for methane monooxygenase encoding genes in 60 currently available Rokubacteriales metagenomes yielded negative results, although copper-containing monooxygenases were encoded by some members of this order. This study suggests that peat-inhabiting Rokubacteriales are neutrophilic non-methanotrophic bacteria that colonize nitrogen-rich wetlands.

Microbial Tyrosinase: Biochemical, Molecular Properties and Pharmaceutical Applications

Tyrosinase is a copper-containing monooxygenase involved in thecatalysis of the hydroxylation and oxidation reaction of monophenols and diphenols, respectively, into O-quinones intermediates. Tyrosinase is mainly involved in melanogenesis via two reactions. Firstly, 3,4-dihydroxyphenylalanine is produced through tyrosine hydroxylation the nit oxidized into dopaquinone, and finally gives melanin. However, dopaquinones can results in neuronal damage and cell death through the excessive production, suggesting that tyrosinase may be implanted in the formation human brain’s neuromelanin and association with Parkinson’s diseases. Thus, down regulating the melanin pigments and its intermediates by inhibiting tyrosinase activity is the major pharmaceutical challenge to prevent hyperpigmentation, in addition to therapy of neuromelanin disorders. Thus, this review has been focused on exploring the biochemical and molecular properties of tyrosinase from different sources and its potential inhibition with different natural and synthetic compounds.

Engineered tyrosinases with broadened bio-catalysis scope: immobilization using nanocarriers and applications.

Enzyme immobilization is a widely used technology for creating more stable, active, and reusable biocatalysts. The immobilization process also improves the enzyme's operating efficiency in industrial applications. Various support matrices have been designed and developed to enhance the biocatalytic efficiency of immobilized enzymes. Given their unique physicochemical attributes, including substantial surface area, rigidity, semi-conductivity, high enzyme loading, hyper catalytic activity, and size-assisted optical properties, nanomaterials have emerged as fascinating matrices for enzyme immobilization. Tyrosinase is a copper-containing monooxygenase that catalyzes the o-hydroxylation of monophenols to catechols and o-quinones. This enzyme possesses a wide range of uses in the medical, biotechnological, and food sectors. This article summarizes an array of nanostructured materials as carrier matrices for tyrosinase immobilization. Following a detailed background overview, various nanomaterials, as immobilization support matrices, including carbon nanotubes (CNTs), carbon dots (CDs), carbon black (CB), nanofibers, Graphene nanocomposite, platinum nanoparticles, nano-sized magnetic particles, lignin nanoparticles, layered double hydroxide (LDH) nanomaterials, gold nanoparticles (AuNPs), and zinc oxide nanoparticles have been discussed. Next, applied perspectives have been spotlights with particular reference to environmental pollutant sensing, phenolic compounds detection, pharmaceutical, and food industry (e.g., cereal processing, dairy processing, and meat processing), along with other miscellaneous applications.

Draft Genome Sequences of Four Aerobic Isobutane-Metabolizing Bacteria.

Here, we report the draft genome sequences of four aerobic gaseous alkane-oxidizing bacteria isolated from soil by enrichment culture using isobutane (2-methylpropane) as the sole carbon and energy source. The sequences all reveal microorganisms with multiple alkane-oxidizing monooxygenases, including soluble di-iron monooxygenases (SDIMOs), copper-containing monooxygenases (CuMMOs), and alkane hydroxylases (AHs).

Biophysical Compatibility of a Heterotrimeric Tyrosinase-TYRP1-TYRP2 Metalloenzyme Complex.

Tyrosinase (TYR) is a copper-containing monooxygenase central to the function of melanocytes. Alterations in its expression or activity contribute to variations in skin, hair and eye color, and underlie a variety of pathogenic pigmentary phenotypes, including several forms of oculocutaneous albinism (OCA). Many of these phenotypes are linked to individual missense mutations causing single nucleotide variants and polymorphisms (SNVs) in TYR. We previously showed that two TYR homologues, TYRP1 and TYRP2, modulate TYR activity and stabilize the TYR protein. Accordingly, to investigate whether TYR, TYRP1, and TYRP2 are biophysically compatible with various heterocomplexes, we computationally docked a high-quality 3D model of TYR to the crystal structure of TYRP1 and to a high-quality 3D model of TYRP2. Remarkably, the resulting TYR-TYRP1 heterodimer was complementary in structure and energy with the TYR-TYRP2 heterodimer, with TYRP1 and TYRP2 docking to different adjacent surfaces on TYR that apposed a third realistic protein interface between TYRP1-TYRP2. Hence, the 3D models are compatible with a heterotrimeric TYR-TYRP1-TYRP2 complex. In addition, this heterotrimeric TYR-TYRP1-TYRP2 positioned the C-terminus of each folded enzymatic domain in an ideal position to allow their C-terminal transmembrane helices to form a putative membrane embedded three-helix bundle. Finally, pathogenic TYR mutations causing OCA1A, which also destabilize TYR biochemically, cluster on an unoccupied protein interface at the periphery of the heterotrimeric complex, suggesting that this may be a docking site for OCA2, an anion channel. Pathogenic OCA2 mutations result in similar phenotypes to those produced by OCA1A TYR mutations. While this complex may be difficult to detect in vitro, due to the complex environment of the vertebrate cellular membranous system, our results support the existence of a heterotrimeric complex in melanogenesis.

Biochemical Properties of Tyrosinase from Aspergillus terreus and Penicillium copticola; Undecanoic Acid from Aspergillus flavus, an Endophyte of Moringa oleifera, Is a Novel Potent Tyrosinase Inhibitor.

Tyrosinase is a copper-containing monooxygenase catalyzing the O-hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine then to dopaquinone that is profoundly involved in melanin synthesis in eukaryotes. Overactivation of tyrosinase is correlated with hyperpigmentation that is metabolically correlated with severe pathological disorders, so, inhibition of this enzyme is the most effective approach in controlling the overproduction of melanin and its hazardous effects. Thus, searching for a powerful, selective inhibitor of human tyrosinase to limit the hyper-synthesis of melanin is a challenge. Unlike the difficulty of overexpression of human tyrosinase, using fungal tyrosinase as a model enzyme to the human one to evaluate the mechanistics of enzyme inhibition in response to various compounds is the most feasible strategy. Thus, the purification of highly catalytic-efficient fungal tyrosinase, exploring a novel inhibitor, and evaluating the mechanistics of enzyme inhibition are the main objectives of this work. Aspergillus terreus and Penicillium copticola were reported as the most potential tyrosinase producers. The biochemical properties suggest that this enzyme displays a higher structural and catalytic proximity to human tyrosinase. Upon nutritional bioprocessing by Plackett–Burman design, the yield of tyrosinase was increased by about 7.5-folds, compared to the control. The purified tyrosinase was strongly inhibited by kojic acid and A. flavus DCM extracts with IC50 values of 15.1 and 12.6 µg/mL, respectively. From the spectroscopic analysis, the main anti-tyrosinase compounds of A. flavus extract was resolved, and verified as undecanoic acid. Further studies are ongoing to unravel the in vivo effect and cytotoxicity of this compound in fungi and human, that could be a novel drug to various diseases associated with hyperpigmentation by melanin.

Dimethylanilinic N-Oxides and Their Oxygen Surrogacy Role in the Formation of a Putative High-Valent Copper-Oxygen Species.

The reaction of p-cyano-N,N-dimethylaniline N-oxide, an O-atom donor, with different copper(I) complexes (at room temperature and in acetone) indicates the formation via O-atom transfer of a high-valent copper oxyl species, CuII-O•, a putative key intermediate in the catalytic cycle of copper-containing monooxygenases. The formation of p-cyano-N-hydroxymethyl-N-methylaniline and p-cyano-N-methylaniline as the main products of the reaction highlight the capability of this species to hydroxylate strong C-H bonds (bond dissociation energy ∼ 90 kcal/mol). A plausible mechanism for the reactivity of this catalytic system is proposed.

Regioselective oxidative carbon-oxygen bond cleavage catalysed by copper(II) complexes: A relevant model study for lytic polysaccharides monooxygenases activity

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing monooxygenase and catalyzing the oxidative cleavage of recalcitrant polysaccharides using dioxygen. The copper(II) complexes [Cu(L1)(H2O)ClO4]ClO41, [L1 = 4-methyl-1-[(pyridin-2-yl-methyl)]-1,4-diazepane]; [Cu(L2)(H2O)ClO4]ClO42, [L2 = 4-methyl-1-[(2-(pyridine-2-yl)ethyl]-1,4-diazepane] and [Cu(L3)(H2O)ClO4]ClO43, [L3 = 1-(4-methoxy-3,5-dimethyl-pyridin-2-yl)methyl)-4-methyl-1,4-diazepane] have been synthesized and characterized as the novel models for LPMOs. The molecular structures exhibit distorted square pyramidal geometry (τ, 0.183–0.388) as similar to LPMOs. The CuN (1.99–2.02 Å) bond distances of the model complexes are almost identical to those of native LPMOs enzyme (1.9–2.2 Å). The 1, 4-diazepane backbone and pyridine unit of complexes provide reasonable structural resemblances to ‘histidine brace’ and histidine residues of LPMOs respectively. The spectral, redox and kinetic studies were performed in water to mimic accurate enzymatic reaction conditions. The well-defined Cu(II)/Cu(I) reduction couples were observed around 8–112 mV versus NHE, which is lower than that of LPMOs. The electronic spectra of the complexes showed the d-d transitions around 600–635 nm and axial EPR parameter (g||, 2.28–2.29; A||, 160–168 × 10−4 cm−1), which are almost identical to that of LPMOs. The model complexes were catalyzed oxidative cleavage of model substrate p-nitrophenyl-β-D-glucopyranoside into p-nitrophenol and D-allose with a maximum yield up to 78.4% and TON, 300. The kinetics of reaction monitored by following the formation of an absorption band around at 400 nm corresponds to p-nitrophenol, showed the rate of 3.19–5.26 × 10−3 s−1. The oxidative cleavage reaction may occur via CuII-OOH intermediate, whose formation accompanied by an electronic spectral signature around 375 nm with the rate of 1.61–9.06 × 10−3 s−1. The CuII-OOH intermediate was characterized by spectral methods and its geometry was optimized by DFT calculations.

Catalytic mechanism of the tyrosinase reaction toward the Tyr98 residue in the caddie protein.

Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a “caddie” protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr98 residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the μ-η2:η2-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now—despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations—the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16–1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of CuA, which induces the syn-to-anti rearrangement of the copper ligands after the formation of μ-η2:η2-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu2O2 oxidant.

Involvement of dopamine beta-hydroxylase in the neuroendocrine-immune regulatory network of white shrimp, Litopenaeus vannamei

In shrimp, the biosynthesis of catecholamines, including dopamine and norepinephrine, is required for physiological and immunological responses against stress. Dopamine beta-hydroxylase (DBH), a copper-containing monooxygenase enzyme that plays an important role in catecholamine synthesis of the neuroendocrine regulatory network, was identified in Litopenaeus vannamei. In the present study, the potential role of DBH in the immunocompetence of L. vannamei was further estimated by depleting DBH by pharmaceutical inhibition of disulfiram and a gene silencing technique of L. vannamei DBH-double-stranded (ds)RNA (LvDBH-dsRNA). Immunocompetence was evaluated following the determination of the total hemocyte count, differential hemocyte count, phenoloxidase activity, respiratory bursts, superoxide dismutase activity, phagocytic activity, and the clearance efficiency as well as the susceptibility against Vibrio alginolyticus infection. At 30–120 min after shrimp had received disulfiram, they exhibited significantly reduced total hemocyte count, phenoloxidase activity of hemocytes in hemolymph, respiratory bursts of hemocytes in hemolymph and per hemocyte, phagocytic activity, clearance efficiency, and survival ratio against V. alginolyticus infection, compared to those injected with saline. In addition, the significantly lower total hemocyte count, phagocytic activity, clearance efficiency, and resistance to V. alginolyticus infection were observed in shrimp that received LvDBH-dsRNA at 3 days post injection compared to those injected with diethyl pyrocarbonate-water or non-targeting gene-dsRNA. The DBH depleted L. vannamei revealed immunosuppression and decreased the survival ratio to V. alginolyticus infection, which indicated that DBH played a crucial role in the neuroendocrine-immune regulatory network.

Aspergillus niger PA2 Tyrosinase Covalently Immobilized on a Novel Eco-Friendly Bio-Composite of Chitosan-Gelatin and Its Evaluation for L-DOPA Production

Tyrosinase (EC 1.14.18.1) a copper-containing monooxygenase, isolated from a fungal isolate Aspergillus niger PA2 was subjected for immobilization onto a composite consisting of chitosan and gelatin biopolymers. The homogeneity of the chitosan-gelatin biocomposite film was characterized by X-ray diffraction analyses. To evaluate immobilization efficiency, chitosan-gelatin-Tyr bio-composite films were analyzed by field emission scanning electron microscopy, atomic force microscopy and UV-spectroscopy. The rough morphology of the film led to a high loading of enzyme and it could retain its bioactivity for a longer period. The enzyme adsorbed onto the film exhibited 72% of its activity after 10 days and exhibited good repeatability for up to nine times, after intermittent storage. Moreover, the immobilized enzyme exhibited broader pH and temperature profile as compared to free counterpart. Immobilized enzyme was further evaluated for the synthesis of L-DOPA (2,4-dihydroxy phenylalanine) which is a precursor of dopamine and a potent drug for the treatment of Parkinson's disease and for myocardium neurogenic injury.

Are Human Tyrosinase and Related Proteins Suitable Targets for Melanoma Therapy?

Among the human copper-containing monooxygenases, Tyrosinase (Ty) is an important enzyme involved in the determinant step of the biosynthetic pathway of melanin pigment. In this pathway, Ty catalyzes the tyrosine monooxygenation into L-DOPA-quinone, which is the precursor of the skin pigment melanin. Ty inhibitors/activators are a well-established approach for controlling in vivo melanin production, so their development has a huge economical and industrial impact. Moreover, recent publications highlight that targeting tyrosinase with inhibitors/activators to treat melanogenesis disorders is one of many possible approaches, due to the complex biochemical reaction involved in the melanin synthesis.

The evolution of the AMOA, PMOA and BMOA genes measured by ka/ks ratios.

The copper-containing membrane-bound monooxygenase enzyme family consists of enzymes facilitating the oxidation of methane (catalyzed by particulate methane monooxygenase, pMMO) and ammonia (catalyzed by ammonia monooxygenase, AMO), in both aerobic and anaerobic methanotrophs and nitrifiers, respectively, and the oxidation of C2-10 alkanes (catalyzed by particulate butane monooxygenase, pBMO). Despite catalyzing different substrates, pMMO, AMO and pBMO form catabolic enzymes related structurally and evolutionarily to each other, and are encoded by the genes pmoA, amoA and bmoA, respectively. It has been suggested that oxygen-dependent methane and ammonia monooxygenases evolved from a substrate-promiscuous ancestor after horizontal transfer(s) into new hosts, which eventually became methanotrophs and nitrifiers. However, no extensive studies have been conducted on molecular adaptation of the beta-peptide structure of pmoA, amoA and bmoA gene products, which form transmembrane polypeptides, and on selective pressures acting on them. Over 80 near-complete pmoA, amoA and bmoA gene from database were downloaded and analyzed to assess whether the combined amoA, pmoA and bmoA gene tree is congruent to the 16S rRNA gene tree for these microbes. The gene was separated into 15 segments and pairwise Ka/Ks values were calculated to assess the evolutionary and selection pressure exerted on these taxa, based on the hypothesis that they evolved from a methanotrophic ancestor, as well as calculate Ka/Ks values between members of each family.Our analyses indicated a high level of negative (or purifying) selection between and amongst all taxa. Ka/Ks values were highest between methanotroph pmoA and the anammox, and that the pmoA and amoA genes are under purifying selection their clusters, and supports the hypothesis that one of these genes likely arose from the other via horizontal gene transfer.

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications.

Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8.

Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments. Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere. Clearly, nitrogen availability in the form of ammonium and nitrite have strong effects on methanotrophic activity and their natural community structures. Previous findings show that nitrite amendment inhibits the activity of some cultivated methanotrophs; however, the physiological pathways that allow some strains to transform nitrite, expression of gene inventories, as well as the electron sources that support this activity remain largely uncharacterized. Here we show that Methylomicrobium album strain BG8 utilizes methane, methanol, formaldehyde, formate, ethane, ethanol, and ammonia to support denitrification activity under hypoxia only in the presence of nitrite. We also demonstrate that transcript abundance of putative denitrification genes, nirS and one of two norB genes, increased in response to nitrite. Furthermore, we found that transcript abundance of pxmA, encoding the alpha subunit of a putative copper-containing monooxygenase, increased in response to both nitrite and hypoxia. Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation. The present study expands current knowledge of the metabolic flexibility of methanotrophs by revealing that a diverse array of electron donors support nitrite reduction to nitrous oxide under hypoxia.

Synthesis and Characterization of a Dinuclear Copper Complex Bearing a Hydrophobic Cavity as a Model for Copper‐Containing Monooxygenases

AbstractThe synthesis and characterization of a dinuclear copper complex as a model of mononuclear copper‐containing monooxygenases [dopamine β‐monooxygenase (DβM), tyramine β‐monooxygenase (TβM), and peptidylglycine α‐hydroxylating monooxygenase (PHM)] are described. This model is based on a cyclophane moiety bearing two bis(picolylamine) ligands for the complexation of two copper atoms and exhibits macroscopic features equivalent to those of the enzymes: (1) the two copper centers are 11.5 Å apart (11 Å for the enzymes) and (2) there is a hydrophobic pocket consisting of a cyclophane moiety in which substrates can bind near the two copper centers.

A copper-containing oxidase catalyzes C-nitrosation in nitrosobenzamide biosynthesis

Although C-nitroso aromatic compounds have several bioactivities of medicinal interest, the biosynthetic enzymes involved in C-nitrosation have remained unknown until now. Here, we report the entire biosynthesis pathway of 4-hydroxy-3-nitrosobenzamide in Streptomyces murayamaensis, in which a tyrosinase-like copper-containing monooxygenase is responsible for the C-nitrosation. This finding indicates diverse catalytic functions of tyrosinase-like copper-containing monooxygenases in nature.

Constituents from the Formosan apple reduce tyrosinase activity in human epidermal melanocytes

Tyrosinase is a copper-containing monooxygenase that catalyzes melanin synthesis in skin melanocytes. Herein, 13 compounds from the Formosan apple ( Malus doumeri var. formosana), an indigenous Taiwanese plant, were isolated and identified. The active constituents were identified as 3-hydroxyphloretin ( 7) and catechol ( 9); they exhibited potent hydroxyl radical-scavenging (IC 50 values, 0.6 and 1.1 μM) and cellular tyrosinase-reducing activities (IC 50 values, 32 and 22 μM) in human epidermal melanocytes. In addition, we evaluated the level of several tyrosinase-related proteins by Western blot analysis. In contrast to 3-hydroxyphloretin ( 7), which showed no effect on the level of these proteins, catechol ( 9) reduced their activity and the expression of the respective genes, as determined by quantitative real-time PCR. In a kinetic analysis of mushroom tyrosinase, 3-hydroxyphloretin ( 7) was a competitive inhibitor. These two constituents exhibited metal-coordinating interactions with copper ions in a virtual model of molecular docking with human tyrosinase. Thus, 3-hydroxyphloretin ( 7) and catechol ( 9) were the most active constituents from the Formosan apple; they exhibited anti-oxidant and tyrosinase reducing activities, suggesting their possible use as cosmetic agents.

The Mechanism of Suicide-Inactivation of Tyrosinase: A Substrate Structure Investigation

Tyrosinase is a copper-containing mono-oxygenase, widely distributed in nature, able to catalyze the oxidation of both phenols and catechols to the corresponding ortho-quinones. Tyrosinase is characterised by a hitherto unexplained irreversible inactivation which occurs during the oxidation of catechols. Although the corresponding catechols are formed during tyrosinase oxidation of monophenols, inactivation in the presence of monophenolic substrates is minimal. Previous studies have established the kinetic features of the inactivation reaction which is first-order in respect of the enzyme concentration. The inactivation reaction exhibits the same pH-profile and saturation properties as the oxidation reaction, classing the process as a mechanism-based suicide inactivation. The recent elucidation of the crystallographic structure of tyrosinase has stimulated a new approach to this long-standing enigma. Here we report the results of an investigation of the tyrosinase-catalysed oxidation of a range of hydroxybenzenes which establish the structural requirements associated with inactivation. We present evidence for an inactivation mechanism based on catechol hydroxylation, with loss of one of the copper atoms at the active site. The inactivation mechanism involves two linked processes occurring in situ: (a) catechol presentation resulting in alpha-oxidation, and (b) deprotonation of an adjacent group. On the basis of our experimental data we believe that a similar mechanism may account for the inhibitory action of resorcinols.

Inhibitory Effects on L-Dopa Oxidation of Tyrosinase by Skin-whitening Agents

Tyrosinase (EC 1.14.18.1) is a copper-containing monooxygenase catalyzing the o-hydroxylation of monophenols to the corresponding catechols (monophenolase or cresolase activity), and the oxidation of monophenols to the corresponding o-quinones (diphenolase or catecholase activity). These functions of tyrosinase play an important role in the formation of melanin pigments during melanogenesis. Melanin production is principally responsible for skin color and plays an important role in prevention of sun-induced skin injury. However, abnormal accumulation of melanin products is responsible for hyperpigmentations including melasma, freckles, and senile lentigines, which could be a serious aesthetic problem. Recently, a global market demand has developed for skinwhitening agents because some dark-skinned individuals in many countries and races are prefer lighter skin color. Several tyrosinase inhibitors have been used in the cosmetic industry as skin-whitening agents. Unfortunately, many of these inhibitors have not been demonstrated yet to be clinically efficacious when critically analyzed in carefully controlled studies. Thus, many efforts are still devoted to screening recognized and putative skin-whitening agents. A correct assay method is essential for the development of effective inhibitors of tyrosinase. Until now, the screening and evaluation methods of these skin-whitening agents have been adopted dopa oxidation test by mushroom tyrosinase because of its commercial availability, not human tyrosinase. Moreover, a comparative study on the inhibitory effects of skin-whitening agents by human tyrosinase and mushroom tyrosinase is still undone. In this study, we investigated inhibitory effects of skinwhitening agents on L-dopa oxidation by human tyrosinase and mushroom tyrosinase, and compared their effects. According to our study described here, L-dopa oxidation activities of human tyrosinase and mushroom tyrosinase show very different inhibitory effects depending upon the nature of skin-whitening agents.