Molecular Docking Studies of Ortho-Substituted Phenols to Tyrosinase Helps Discern If a Molecule Can Be an Enzyme Substrate.

Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring.

Salvia officinalis L. Essential Oils from Spain: Determination of Composition, Antioxidant Capacity, Antienzymatic, and Antimicrobial Bioactivities.

Four essential oils (EOs) from Salvia officinalis L. cultivated in Spain (Murcia Province) were analyzed by gas chromatography coupled with mass spectrometry (GC/MS) to determine their relative and absolute compositions. The main components were α-thujone (22.8 - 41.7%), camphor (10.7 - 19.8%), 1,8-cineole (4.7 - 15.6%), and ß-thujone (6.1 - 15.6%). Enantioselective gas chromatography identified (-)-α-thujone and (+)-camphor as the main enantiomers in all the analyzed EOs. Furthermore, when the EOs were tested to determine their antioxidant activity against free radicals and as ferric reducing and ferrous chelating agents, all were seen to have moderate activity due to the compounds they contained, such as linalool or terpinene. Because of their known relation with inflammatory illnesses and Alzheimer's disease, respectively, the inhibition of lipoxygenase and acetylcholinesterase was studied using the EOs. Some individual compounds also inhibited these enzymes. In addition, the studied EOs were able to inhibit the growth of Escherichia coli, Staphylococcus aureus, and Candida albicans. The characterization carried out increases our awareness of the possible uses of S. officinalis EO as natural additives in food, cosmetics, and pharmaceuticals.

Composition and Antioxidant, Antienzymatic and Antimicrobial Activities of Volatile Molecules from Spanish Salvia lavandulifolia (Vahl) Essential Oils.

The current study describes the composition of Salvia lavandulifolia (Vahl) essential oils (SlEOs) obtained from plants cultivated in Murcia (Spain), as determined by gas chromatography. Relative and absolute concentrations, the enantiomeric ratios of chiral compounds and the in vitro antioxidant, antienzymatic and antimicrobial activities are described. The main components of the SlEOs were camphor, 1,8-cineole, camphene and α-pinene, and the main enantiomers were (+)-camphor and (-)-camphene. The activities against free radicals and the capacity to reduce and chelate metallic ions were measured. SlEO-3 showed the highest activity in ORAC, DPPH, ABTS and reducing power methods, while SlEO-1 exhibited the highest chelating power. The activity of lipoxygenase and acetylcholinesterase could be inhibited by all the SlEOs, being bornyl acetate and limonene the most active individual compounds against lipoxygenase and 1,8-cineole against acetylcholinesterase. SlEOs and some individual compounds inhibited Escherichia coli, Staphylococcus aureus and Candida albicans. These results increase our knowledge of SlEOs and, particularly, provide for the first time a complete characterization of SlEOs from Murcia, Spain, while proposing possible biotechnological uses for them.

4-n-butylresorcinol, a depigmenting agent used in cosmetics, reacts with tyrosinase.

4-n-Butylresorcinol (BR) is considered the most potent inhibitor of tyrosinase, which is why it is used in cosmetics as a depigmenting agent. However, this work demonstrates that BR is a substrate of this enzyme. The Em (met-tyrosinase) form is not active on BR, but Eox (oxy-tyrosinase) can act on this molecule, hydroxylating it to o-diphenol. In turn, this is oxidized to an o-quinone, which isomerizes to a red p-quinone. Thus, for tyrosinase to act on this compound, a mechanism to generate Eox in the medium is required, which can be achieved by means of hydrogen peroxide or ascorbic acid. A kinetic analysis of the proposed mechanism allows its kinetic characterization: catalytic constant kcatBR (8.49 ± 0.20 s(-1) ) and Michaelis-constant KMBR (60.26 ± 8.76 µM). These findings are compared with those for other monophenolic substrates of tyrosinase. Studies of BR docking to the Em form of the enzyme show that the hydroxyl group in C-1 position is oriented toward the copper atom A (CuA), as in it is L-tyrosine. As regards Eox , BR is oriented with the carbon in C-6 position ready to be hydroxylated. The reaction of BR originates o-quinones, which isomerize to p-quinones, which in turn, could react with thiol compounds, a finding that could have important implications for pharmacology and the cosmetic industry. © 2016 IUBMB Life, 68(8):663-672, 2016.

Characterization of the action of tyrosinase on resorcinols.

The action of tyrosinase on resorcinol and some derivatives (4-ethylresorcinol, 2-methylresorcinol and 4-methylresorcinol) was investigated. If the catalytic cycle is completed with a reductant such as ascorbic acid or an o-diphenol such as 4-tert-butylcatechol, these compounds act as substrates of tyrosinase in all cases. The reaction can also be carried out, adding hydrogen peroxide to the medium. All the above compounds were characterized as substrates of the enzyme and their kinetic constants, KM (Michaelis constant) and kcat (catalytic constant) were determined. Measurement of the activity of the enzyme after pre-incubation with resorcinol, 4-ethylresorcinol or 4-methylresorcinol points to an apparent loss of activity at short times, which could correspond to an enzymatic inactivation process. However, if the measurements are extended over longer times, a burst is observed and the enzymatic activity is recovered, demonstrating that these compounds are not suicide substrates of the enzyme. These effects are not observed with 2-methylresorcinol. The docking results indicate that the binding of met-tyrosinase with these resorcinols occurs in the same way, but not with 2-methylresorcinol, due to steric hindrance.

Lavandula angustifolia and Lavandula latifolia Essential Oils from Spain: Aromatic Profile and Bioactivities.

Compositions of true lavender (Lavandula angustifolia) and spike lavender (Lavandula latifolia) essential oils, cultivated and extracted in the Southeast of Spain, were determined by gas chromatography coupled with mass spectrometry detection, obtaining both relative (peak area) and absolute (using standard curves) concentrations. Linalool (37-54 %), linalyl acetate (21-36 %) and (E)-ß-caryophyllene (1-3 %) were the most abundant components for L. angustifolia. Linalool (35-51 %), eucalyptol (26-32 %), camphor (10-18 %), α-pinene (1-2 %), α-terpineol (1-2 %) and α-bisabolene (1-2 %) were the most abundant components for L. latifolia. The characterization was completed with enantioselective gas chromatography, in which the determined main molecules were (-)-linalool, (-)-linalyl acetate and (+)-camphor. (S)-(-)-camphene, (R)-(+)-limonene, (1R, 9S)-(-)-(E)-ß-caryophyllene and (1R, 4R, 6R, 10S)-(-)-caryophyllene oxide were found in this study as the predominant enantiomers in Spanish L. angustifolia. The characterised essential oils were tested for their antioxidant activity against free radicals ABTS, DPPH, ORAC, chelating, and reducing power. Inhibitory activity on lipoxygenase was observed indicating a possible anti-inflammatory activity, mainly due to linalool, camphor, p-cymene and limonene. These results can be the starting point for a future study of the potential use of L. angustifolia and L. latifolia essential oils as natural cosmetic and natural pharmaceutical ingredients for several skin diseases.

Kinetic characterization of oxyresveratrol as a tyrosinase substrate.

Oxyresveratrol is a stilbenoid described as a powerful inhibitor of tyrosinase and proposed as skin-whitening and anti-browning agent. However, the enzyme is capable of acting on it, considering it as a substrate, as it has been proved in the case of its analogous resveratrol. Tyrosinase hydroxylates the oxyresveratrol to an o-diphenol and oxidizes the latter to an o-quinone, which finally isomerizes to p-quinone. For these reactions to take place the presence of the Eox (oxy-tyrosinase) form is necessary. The kinetic analysis of the proposed mechanism has allowed the kinetic characterization of this molecule as a substrate of tyrosinase, affording a catalytic constant of 5.39 ± 0.21 sec(-1) and a Michaelis constant of 8.65 ± 0.73 µM.

Kinetic characterization of substrate-analogous inhibitors of tyrosinase.

The development of effective tyrosinase inhibitors has become increasingly important in the cosmetic, medicinal, and agricultural industries for application as antibrowning and depigmenting agents. The kinetic mechanisms of action of tyrosinase on monophenols and o-diphenols are complex, particularly in the case of monophenols because of the lag period that occurs at the beginning of the reaction. When enzyme inhibitors are studied, the problem becomes more complicated because the lag period increases, which has led to erroneous identification of the type of inhibition that many compounds exert on the monophenolase activity and the inaccurate determination of their inhibition constants. When the degrees of inhibition of an inhibitor which is analogous to tyrosinase substrates are the same for both monophenolase and diphenolase activities, this means that the inhibitor binds to the same enzymatic species and so the inhibition constants should be similar for both activities. In this study, we demonstrate this typical behavior of substrate-analogous inhibitors and propose a methodology for determining the type of inhibition and the inhibition constants for the monophenolase and diphenolase activities of the enzyme. Benzoic acid and cinnamic acid were used as inhibitors and the monophenol/o-diphenol pairs l-tyrosine/l-dopa and α-methyl-L-tyrosine/α-methyl-L-dopa as substrates.

Identification of p-hydroxybenzyl alcohol, tyrosol, phloretin and its derivate phloridzin as tyrosinase substrates.

In recent years, the hydroxyalkylphenols p-hydroxybenzyl alcohol and tyrosol, and the compound phloretin and its derivate phloridzin have been described as inhibitors of the enzyme tyrosinase. When the monophenolase and the diphenolase activities of tyrosinase on its physiological substrates l-dopa and/or l-tyrosine are measured in the presence of these compounds, the rate of action of the enzyme decreases. These findings led to the identification of these compounds as inhibitors. However, these molecules show an unusual behavior as inhibitors of the enzyme indeed, in this study, we demonstrate that they are not true inhibitors but alternative substrates of the enzyme.

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase.

We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamineso-aminophenols>aromatic o-diamines.

A tumor-associated heparan sulfate-related glycosaminoglycan promotes the generation of functional regulatory T cells.

Functional Tregs play a key role in tumor development and progression, representing a major barrier to anticancer immunity. The mechanisms by which Tregs are generated in cancer and the influence of the tumor microenvironment on these processes remain incompletely understood. Herein, by using NMR, chemoenzymatic structural assays and a plethora of in vitro and in vivo functional analyses, we demonstrate that the tumoral carbohydrate A10 (Ca10), a cell-surface carbohydrate derived from Ehrlich's tumor (ET) cells, is a heparan sulfate-related proteoglycan that enhances glycolysis and promotes the development of tolerogenic features in human DCs. Ca10-stimulated human DCs generate highly suppressive Tregs by mechanisms partially dependent on metabolic reprogramming, PD-L1, IL-10, and IDO. Ca10 also reprograms the differentiation of human monocytes into DCs with tolerogenic features. In solid ET-bearing mice, we found positive correlations between Ca10 serum levels, tumor size and splenic Treg numbers. Administration of isolated Ca10 also increases the proportion of splenic Tregs in tumor-free mice. Remarkably, we provide evidence supporting the presence of a circulating human Ca10 counterpart (Ca10H) and show, for the first time, that serum levels of Ca10H are increased in patients suffering from different cancer types compared to healthy individuals. Of note, these levels are higher in prostate cancer patients with bone metastases than in prostate cancer patients without metastases. Collectively, we reveal novel molecular mechanisms by which heparan sulfate-related structures associated with tumor cells promote the generation of functional Tregs in cancer. The discovery of this novel structural-functional relationship may open new avenues of research with important clinical implications in cancer treatment.

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase.

The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding o-diphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.

Suicide inactivation of tyrosinase in its action on tetrahydropterines.

Tetrahydrobiopterin (BH(4)), methyl-tetrahydropterin (MBH(4)) and dimethyl-tetrahydropterin (DMBH(4)) are oxidized by tyrosinase in a process during which the suicide inactivation of tyrosinase may occur. From the kinetic study of this process, [Formula: see text] (apparent maximum constant for the suicide inactivation), [Formula: see text] (Michaelis constant for the substrate) and r (number of turnovers that the enzyme makes before the inactivation) can be obtained. From the results obtained, it can be deduced that the velocity of the inactivation governed by ([Formula: see text]) and the potency of the same ([Formula: see text]) follow the order: BH(4) > MBH(4) > DMBH(4).

Update on fungemia in oncology and hematology.

The present article is an update of the literature on fungemia in onco-hematologic patients. A multidisciplinary group of Spanish physicians with an interest in this field selected the most important papers published lately. Papers from the fields of epidemiology, risk factors, pathogenesis, diagnosis, outcome, prevention and treatment are discussed. Important aspects of these studies include the assessment of different strategies in the management of fever in neutropenic patients. Moreover, early identification of patients at risk of fungal infections, as well as identification of patients at risk for fluconazole-resistant strains are topics of increasing interest.

In vitro neuroprotective potential of terpenes from industrial orange juice by-products.

Citrus sinensis (orange) by-products represent one of the most abundant citric residues from orange juice industrial production, and are a promising source of health-promoting compounds like terpenes. In this work, different extraction solvents have been employed to increase terpene extraction yield and selectivity from this orange juice by-product. A set of bioactivity assays including enzymatic (acetylcholinesterase (AChE), butylcholinesterase (BChE) and lipoxygenase (LOX)) as well as antioxidant (ABTS, reactive oxygen species (ROS) and reactive nitrogen species (RNS)) activity tests have been applied to investigate the neuroprotective potential of these compounds. New fluorescence-based methodologies were developed for AChE and BChE assays to overcome the drawbacks of these tests when used in vitro to determine the anticholinergic activity of colored extracts. Comprehensive phytochemical profiling based on gas chromatography coupled to quadrupole time of flight mass spectrometry (GC-qTOF-MS) analysis showed ahigh content of mono- and sesquiterpenes in the extracts obtained with ethyl acetate, whereas n-heptane extracts exhibited a large amount of triterpenes and carotenoids. From a neuroprotective activity point of view, ethyl acetate extract is the most promising due to its anticholinergic activity and antioxidant capacity. Finally, a multivariate data analysis revealed a good correlation between some monoterpenes (e.g. nerol or limonene) and the antioxidant capacity of the natural extract, while a group of sesquiterpenes (e.g.δ-Cadinene or nootkatone) showed correlation with the observed AChE, BChE and LOX inhibition capacity. Hydrocarbons mono- and sesquiterpenoids reveal high capacity in vitro to cross the blood-brain barrier (BBB).

Generation of hydrogen peroxide in the melanin biosynthesis pathway.

The generation of H(2)O(2) in the melanin biosynthesis pathway is of great importance because of its great cytotoxic capacity. However, there is controversy concerning the way in which H(2)O(2) is generated in this pathway. In this work we demonstrate that it is generated in a series of chemical reactions coupled to the enzymatic formation of o-quinones by tyrosinase acting on monophenols and o-diphenols and during the auto-oxidation of the o-diphenols and other intermediates in the pathway. The use of the enzymes such as catalase, superoxide dismutase and peroxidase helps reveal the H(2)O(2) generated. Based on the results obtained, we propose a scheme of enzymatic and non-enzymatic reactions that lead to the biosynthesis of melanins, which explains the formation of H(2)O(2).

Stereospecific inactivation of tyrosinase by L- and D-ascorbic acid.

A kinetic study of the inactivation of tyrosinase by L- and D-ascorbic acid isomers has been carried out. In aerobic conditions, a suicide inactivation mechanism operates, which was attributed to the enzymatic form oxytyrosinase. This suicide inactivation is stereospecific as regards the affinity of the enzyme for the substrate but not as regards the speed of the process, which is the same for both isomers, reflecting the influence of the chemical shift of the carbon C-2 (delta(2)) and C-3 (delta(3)) as seen by (13)C-NMR. The inactivation of deoxytyrosinase and mettyrosinase observed in anaerobic conditions, is irreversible and faster than the suicide inactivation process, underlining the fact that the presence of oxygen protects the enzyme against inactivation.

Suicide inactivation of the diphenolase and monophenolase activities of tyrosinase.

The suicide inactivation mechanism of tyrosinase acting on its phenolic substrates has been studied. Kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone versus time. The electronic, steric, and hydrophobic effects of the phenolic substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain this suicide inactivation, we propose a mechanism in which the enzymatic form oxy-tyrosinase is responsible for the inactivation. In this mechanism, the rate constant of the reaction would be directly related with the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the coplanarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction of one copper atom, giving rise to copper (0), hydrogen peroxide, and an o-quinone, which would be released, thus inactivating the enzyme. One possible application of this property could be the use of these suicide substrates as skin depigmenting agents.

Ellagic acid: characterization as substrate of polyphenol oxidase.

Ellagic acid has been described as an inhibitor of tyrosinase or polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that ellagic acid is not an inhibitor, but a substrate of mushroom polyphenol oxidase, an enzyme which oxidizes ellagic acid, generating its o-quinone. Because o-quinones are very unstable, we used an oxymetric method to characterize the kinetics of this substrate, based on measurements of the oxygen consumed in the tyrosinase reaction. The catalytic constant is very low at both pH values used in this work (4.5 and 7.0), which means that the Michaelis constant for the oxygen is low. The affinity of the enzyme for the substrate is high (low K(m) (S)), showing the double possibility of binding the substrate. Moreover, a new enzymatic method is applied for determining the antioxidant activity. Ellagic acid shows high antioxidant activity (EC50 = 0.05; number of electrons consumed by molecule of antioxidant = 10), probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals.