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Scheme of the coordination mode for met-Ty, met-Ty-Kojic acid and met-Ty-chloride complexes according to our MXAN analysis.
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The key structural features that define the reaction mechanism of the binuclear copper enzyme Tyrosinase (Ty) from Streptomyces antibioticus were investigated by X-ray absorption spectroscopy. The data for the met form, the halide bound derivative and the adduct with the competitive inhibitor and transition state analogue Kojic acid were analysed u...
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Context 1
... binding of Kojic acid did not modify the overall structure of the site as can be deduced from our fits (see Table 1). The structural model proposed here (see Fig. 4, top panel) for the Ty-Kojic acid adduct is substantiated by different experimental data present in the literature. Pre- vious EXAFS work by Woolery [16] indicates the same Cu-Cu distance reported in this study for both the met and the L-Mimosine adduct (a high affinity competitive inhibitor of Tyrosinase structurally similar to Kojic ...
Context 2
... structure that it is suggested to resemble the binding mode of the substrate [7]. Further evidence comes from the structural model proposed for the half- met-Ty Kojic acid complex on the basis of the ESEEM (electron spin echo envelope modulation) [10] and HYSCORE [11] that suggest a five coordinated paramag- netic center. The scheme presented in Fig. 4 represents a further contribution in the description of the reaction mechanism of Ty since the bound Kojic acid should resem- ble expected coordination mode of the diphenol substrate g 2 :g 1 didentate bridging on its way to product ...
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The dinuclear copper enzyme tyrosinase (Ty) from genetically engineered Streptomyces antibioticus has been investigated in its paramagnetic half-met form [Cu(I)-Cu(II)]. The cw EPR, pulsed EPR, and hyperfine sublevel correlation spectroscopy (HYSCORE) experiments on the half-met-Ty and on its complexes with three different types of competitive inhi...
This paper shows for the first time that the spectral features of the ternary complex of tyrosinase/O2/phenol, trapped at low temperature using the very slow substrate 3,5-difluorophenol, are those of a mu-eta2:eta2-peroxidodicopper(II) species, and that this remains the only enzyme species under turnover and substrate saturation conditions.
The (1)H NMR relaxation characteristics of the histidines in the oxidised type-3 copper site of tyrosinase (Ty(met)) from the bacterium Streptomyces antibioticus in the halide-bound forms (Ty(met)X with X = F(-), Cl(-), Br(-)) have been determined and analysed. The (1)H NMR spectra of the Ty(met)X species display remarkably sharp, well-resolved, pa...
Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyr...
A fluorescence-based system to sense oxygen in solution is described. The method exploits the sensitivity of the endogenous fluorescence of type-3 copper proteins towards the presence of oxygen by translating the near-UV emission of the protein to label fluorescence in the visible range through a FRET mechanism. The main protein in this study, a re...
Citations
... Subsequent reports by Plenge et al. [93] and Ackermann et al. (2002) [94] put forward the concept of bridging and unsymmetric binding of catechol substrates in a Z2:Z1 fashion, with one of the two oxygen atoms participating in a weak interaction with either of the neighboring copper(II) ions. Studies involving electron spin echoed envelope modulation (ESEEM) [95] and X-ray absorption spectroscopy (XAS) [96] of a met Ty-KA adduct from bacterial Streptomyces antibioticus Ty, providing further support for this binding mechanism. However, when the X-ray structure of the adduct of KA with the met form of Bacillus megaterium Ty was examined, it revealed that the KA molecule was situated at a distance of 7 Å from the dicopper center. ...
... Subsequent reports by Plenge et al. [93] and Ackermann et al. (2002)[94] put forward the concept of bridging and unsymmetric binding of catechol substrates in a Z2:Z1 fashion, with one of the two oxygen atoms participating in a weak interaction with either of the neighboring copper(II) ions. Studies involving electron spin echoed envelope modulation (ESEEM)[95] and X-ray absorption spectroscopy (XAS)[96] of a met Ty-KA adduct ...
Kojic acid (KA) has emerged as a prominent tyrosinase inhibitor with considerable potential in cosmetic applications; however, its susceptibility to instability during storage poses a challenge to its widespread use. This review explores the advancements in addressing this limitation through the development of various KA derivatives, focusing on the modification of the C-7 hydroxyl group. Strategies such as esterification, hydroxy-phenyl ether formation, glycosylation, and incorporation into amino acid or tripeptide derivatives have been employed to enhance stability and efficacy. Among these derivatives, Kojic Acid Dipalmitate (KDP), a palmitic ester derivative of KA, stands out for its notable improvements in stability, permeability, and low toxicity. Recent developments indicate a growing utilization of KDP in cosmetic formulations, with over 132 available products on the market, encompassing various formulations. Formulations based on nanotechnology, which incorporate KDP, have been provided, including nanosomes, nanocreams, multiple emulsions, liposomes, solid lipid nanoparticles (SLNs), ethosomes, and nanoemulsions. Additionally, three patents and seven advanced system deliveries of KDP further underscore its significance. Despite its increasing prevalence, the literature on KDP remains limited. This review aims to bridge this gap by providing insights into the synthesis process, physicochemical properties, pharmaceutical preparation, diverse applications of KDP in cosmetic products, and recent nanotechnology formulations of KDP. This review paper seeks to explore the recent developments in the use of KDP in cosmetics. The goal is to enhance stability, permeability, and reduce the toxicity of KA, with the intention of promoting future research in this promising sector.
... Subsequent reports by Plenge et al. [76] and Ackermann et al. (2002) [77] put forward the concept of bridging and unsymmetric binding of catechol substrates in a Z2:Z1 fashion, with one of the two oxygen atoms participating in a weak interaction with either of the neighboring copper(II) ions. Studies involving electron spin echo envelope modulation (ESEEM) [78] and X-ray absorption spectroscopy (XAS) [79] of a met Ty-KA adduct from bacterial Streptomyces antibioticus Ty provided further support for this binding mechanism. However, when the X-ray structure of the adduct of KA with the met form of Bacillus megaterium Ty was examined, it revealed that the KA molecule was situated at a distance of 7 Å from the dicopper center. ...
Kojic acid (KA) has emerged as a prominent tyrosinase inhibitor with considerable potential in cosmetic applications; however, its susceptibility to instability during storage poses a challenge to its widespread use. This review explores the advancements in addressing this limitation through the development of various KA derivatives, focusing on the modification of the C-7 hydroxyl group. Strategies such as esterification, hydroxy-phenyl ether formation, glycosylation, and incorporation into amino acid or tripeptide derivatives have been employed to enhance stability and efficacy. Among these derivatives, Kojic Acid Dipalmitate (KDP), a palmitic ester derivative of KA, stands out for its notable improvements in stability, permeability, and low toxicity. Recent developments indicate a growing utilization of KDP in cosmetic formulations, with over 132 available products on the market, encompassing various formulations. Additionally, three patents and seven advanced system deliveries of KDP further underscore its significance. Despite its increasing prevalence, the literature on KDP remains limited. This comprehensive review aims to bridge this gap by providing insights into the synthesis process, physicochemical properties, pharmaceutical preparation, diverse applications of KDP in cosmetic products, and recent nanotechnology formulations of KDP. This review paper seeks to explore the recent developments in the use of KDP in cosmetics. The goal is to enhance stability, permeability, and reduce the toxicity of KA, with the intention of promoting future research in this promising sector.
... [20] Additionally, several reports on catechol-like inhibitors (KA and HOPNO) mention that some of them act as mixed inhibitors. [21] This suggests the presence of two different binding sites for these inhibitors: 1) a KA/HOPNO binding in the active site which competes with the substrate (for KA this is experimentally supported by Electron spin echo envelope modulation (ESEEM) [22] , X-ray absorption spectroscopy (XAS) [23] and paramagnetic NMR studies on bacterial Streptomyces antibioticus [24] ) and 2) another site which does not affect the binding of the substrate but inhibits the enzymatic reaction. These propositions were recently confirmed by the Fishman group, [3a, 21d] who has resolved the crystal structures of the Bacillus megaterium Ty (TyBm) in complex with KA. ...
Tyrosinase enzymes (Tys) are involved in the key steps of melanin (protective pigments) biosynthesis and molecules targeting the binuclear copper active site on tyrosinases represent a relevant strategy to regulate enzyme activities. In this work, the possible synergic effect generated by a combination of known inhibitors is studied. For this, derivatives containing kojic acid (KA) and 2‐hydroxypyridine‐N‐oxide (HOPNO) combined with a thiosemicarbazone (TSC) moiety were synthetized. Their inhibition activities were evaluated on purified tyrosinases from different sources (mushroom, bacterial, and human) as well as on melanin production by lysates from the human melanoma MNT‐1 cell line. Results showed significant enhancement of the inhibitory effects compared with the parent compounds, in particular for HOPNO‐TSC. To elucidate the interaction mode with the dicopper(II) active site, binding studies with a tyrosinase bio‐inspired model of the dicopper(II) center were investigated. The structure of the isolated adduct between one ditopic inhibitor (KA‐TSC) and the model complex reveals that the binding to a dicopper center can occur with both chelating sites. Computational studies on model complexes and docking studies on enzymes led to the identification of KA and HOPNO moieties as interacting groups with the dicopper active site.
... The η 2 -semiquinone-bound half-met form may be formed via the η 2 :η 1 -catecholate-bound met form with a one-electron reduction of Cu B . The η 2 :η 1 -catecholate-bound dicopper(II) was previously proposed as an intermediate of the tyrosinase reaction [35][36][37][48][49][50]. However, if the η 2 :η 1 -catecholatebound intermediate was formed in our system, the ring of the caddie Tyr 98 would become unparallel to the ring of the tyrosinase His 194 , generating the steric hindrance. ...
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.
... KA adopts a η 2 :η 1 coordination mode to the dicopper center (Fig. 5A). This η 2 :η 1 coordination mode exhibits strong similarities between Ty-KA model obtained using XAS [129] or NMR spectroscopy [130] (Fig. 5B). The location of KA in BmTy could be an intermediate binding site. ...
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.
... Therefore, 3-HC accommodates within the MT active site in a manner quite similar to that proposed for mimosine 41 and kojic acid. 42 With these inhibitors, which are transition state analogues, the oxygen of the hydroxyl group acted as a bridge between the two cupric ions, thus displacing the pre-existing water (or hydroxide) already present in the met form. As a consequence, a stable adduct between the dicupric cluster and the inhibitor molecule is formed, preventing the approach of the substrate to the catalytic site. ...
The structure-activity relationships of four hydroxycoumarins, two with the hydroxyl group on the aromatic ring of the molecule and two with the hydroxyl group replacing hydrogen of the pyrone ring, and their interactions with mushroom tyrosinase were studied. These compounds displayed different behaviors upon action of the enzyme. The two compounds, ar-hydroxylated 6-hydroxycoumarin and 7-hydroxycoumarin, were both weak substrates of the enzyme. Interestingly, in both cases, the product of the catalysis was the 6,7-hydroxycoumarin, although 5,6- and 7,8-isomers could also theoretically be formed. Additionally, both were able to reduce the formation of dopachrome when tyrosinase acted on its typical substrate, l-tyrosine. Although none of the compounds that contained a hydroxyl group on the pyrone ring were substrates of tyrosinase, the 3-hydroxycoumarin was a potent inhibitor of the enzyme, and the 4-hydroxycoumarin was not an inhibitor. These results were compared with those obtained by in silico molecular docking predictions to obtain potentially useful information for the synthesis of new coumarin-based inhibitors that resemble the structure of the 3-hydroxycoumarin.
... X-ray absorption spectroscopy (XAS), paramagnetic NMR spectroscopy, and electron spin echo envelope modulation (ESEEM) studies suggest direct binding to the copper ions for KA in the adduct with bacterial Streptomyces antibioticus Ty, with a m-h 2 :h 1 binding mode reported. [11] On the other hand, the X-ray crystal structure of the bacterial Bacillus megaterium Ty with KA as a ligand reveals that the KA is not bound to the dicopper(II) center but is located at a distance of 7 from it. [2b] Recently, the crystal structure of Agaricus bisporus mushroom Ty (deoxy state) in complex with tropolone, [12] another TS-analogue inhibitor, revealed that tropolone binds near the binuclear copper site without directly coordinating the copper ions. ...
... As a result, the coordination mode of the bound OPNO ligand resembles the expected coordination mode of the diphenol substrate on its way to product formation [1b] or the proposed model from XAS studies of the Kojic acid adduct with S. antibioticus tyrosinase. [11] Solution studies: The ESI-MS spectrum of the 3-OPNO (10) adduct provides two signals at m/z 894 (z = 1) and 396. 5 Figure S2 in the Supporting Information). These data evidence that the dibridged dimeric copper(II) structure remains (in part) in solution, as already observed for adduct 2-OPNO. ...
The deciphering of the binding mode of tyrosinase (Ty) inhibitors is essential to understand how to regulate the tyrosinase activity. In this paper, by combining experimental and theoretical methods, we studied an unsymmetrical tyrosinase functional model and its interaction with 2-hydroxypyridine-N-oxide (HOPNO), a new and efficient competitive inhibitor for bacterial Ty. The tyrosinase model was a dinuclear copper complex bridged by a chelated ring with two different complexing arms (namely (bis(2-ethylpyridyl)amino)methyl and (bis(2-methylpyridyl)amino)methyl). The geometrical asymmetry of the complex induces an unsymmetrical binding of HOPNO. Comparisons have been made with the binding modes obtained on similar symmetrical complexes. Finally, by using quantum mechanics/molecular mechanics (QM/MM) calculations, we studied the binding mode in tyrosinase from a bacterial source. A new unsymmetrical binding mode was obtained, which was linked to the second coordination sphere of the enzyme.
... An earlier EXAFS study on the met form of fungal tyrosinase suggests that the Cu-Cu distance is 3.4 Å (30), similar to the met2 form in this study. Furthermore, a previous x-ray absorption study on the met form of S. antibioticus tyrosinase (31), which is 82% identical to the S. castaneoglobisporus tyrosinase in terms of the amino acid sequence, suggests that two coppers lie about 3.4 Å apart with two bridging oxygens. The dihydroxo-bridged dicopper(II) center found in our present study may be a characteristic geometry of the met form of tyrosinase. ...
The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated "caddie," which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16-1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.
... Scheme 1 represents a further contribution in the description of the reaction mechanism of Ty, because HOPNO should resemble the expected coordination mode of the catechol substrate on its way to product formation. These results, which are of biological relevance in the context of Ty mechanism, has to be placed in the perspective of the work of Woolery et al. with the L-mimosine as adduct of Neurospora crassa Ty 24 and the more recent one of Bubacco et al. with kojic acid as adduct of Streptomyces antibioticus Ty. 25 In this later study, on the basis of an X-ray absorption analysis, 25 ESEEM, 26 and HYSCORE, 27 kojic acid was proposed to be a bidentate ligand to CuB of Ty with one O-atom axially coordinated and the second probably bridging the two copper ions in a η 2 :η 1 bidentate mode. These finding are of great interest in relation with new Ty inhibitor design. ...
... Scheme 1 represents a further contribution in the description of the reaction mechanism of Ty, because HOPNO should resemble the expected coordination mode of the catechol substrate on its way to product formation. These results, which are of biological relevance in the context of Ty mechanism, has to be placed in the perspective of the work of Woolery et al. with the L-mimosine as adduct of Neurospora crassa Ty 24 and the more recent one of Bubacco et al. with kojic acid as adduct of Streptomyces antibioticus Ty. 25 In this later study, on the basis of an X-ray absorption analysis, 25 ESEEM, 26 and HYSCORE, 27 kojic acid was proposed to be a bidentate ligand to CuB of Ty with one O-atom axially coordinated and the second probably bridging the two copper ions in a η 2 :η 1 bidentate mode. These finding are of great interest in relation with new Ty inhibitor design. ...
2-Hydroxypyridine-N-oxide (HOPNO) is described as a new and efficient transition-state analog (TS-analog) inhibitor for the mushroom tyrosinase with an IC(50) = 1.16 microM and a K(I) = 1.8 microM. Using the binuclear copper(II) complex [Cu(2)(BPMP)(mu-OH)](ClO(4))(2) (2) known as a functional model for the tyrosinase catecholase activity, we isolated and fully characterized a 1:1 (2)/OPNO adduct in which the HOPNO is deprotonated and chelates only one Cu-atom of the binuclear site in a bidentate mode. On the basis of these results, a structural model for the tyrosinase inhibition by HOPNO is proposed.
Human tyrosinase is the first enzyme of the multistep process of melanogenesis. It catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine and the following oxidation of o-diphenol to the corresponding quinone, L-dopaquinone. In spite of its biomedical relevance, its reactivity is far from being fully understood, mostly because of the lack of a suitable expression system. Indeed, until now, studies on substrates and inhibitors of tyrosinases have been performed in vitro almost exclusively using mushroom or bacterial enzymes. We report on the production of a recombinant human tyrosinase in insect cells (Sf9 line). Engineering the protein, improving cell culture conditions, and setting a suitable purification protocol optimized product yield. The obtained active enzyme was truthfully characterized with a number of substrate and inhibitor molecules. These results were compared to those gained from a parallel analysis of the bacterial (Streptomyces antibioticus) enzyme and those acquired from the literature for mushroom tyrosinase, showing that the reactivity of the human enzyme appears unique and pointing out the great bias introduced when using non-human tyrosinases to measure the inhibitory efficacy of new molecules. The described enzyme is therefore an indispensable paradigm in testing pharmaceutical or cosmetic agents addressing tyrosinase activity.
