Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Comparison of substrate specificity of tyrosinases from Trichoderma reesei and Agaricus bisporus

Understanding the substrate specificity of tyrosinases (EC 1.14.18.1) as well as their capability to oxidize peptide-bound tyrosine residues is important in a view of applicability of tyrosinases. In the present study, two fungal tyrosinases, an extracellular enzyme from the filamentous fungus Trichoderma reesei (TrT) and an intracellular enzyme from the edible mushroom Agaricus bisporus (AbT) were compared. Oxidation of various mono- and diphenolic compounds and tyrosine-containing tripeptides was examined and kinetic constants determined using spectrophotometric and oxygen consumption measurements. TrT and AbT were found to show notable differences in their substrate specificity. TrT generally showed 10-fold higher K_m values than AbT. The presence of a carboxylic and amine group in the substrate influenced the enzymes differently. While the substrates with a carboxyl group were observed not to be effectively oxidized by AbT, the amine group seemed to hider the oxidation in the TrT-catalyzed reactions. Moreover, the UV–visible absorption spectra on the oxidation of catechol and hydrocaffeic acid showed that the product patterns were different between the enzymes. The result is interesting as the primary products from tyrosinase-catalyzed reactions were assumed to be identical with both enzymes. Furthermore, a nucleophilic 3-methyl-2-benzothiazolinone hydrazone (MBTH) affected differently on the activity of the tyrosinases: the lag period related to the oxidation of monophenols was prolonged by MBTH with TrT, whereas with AbT the lag was shortened.     

Comparison of the characteristics of fungal and plant tyrosinases

Enzymatic crosslinking provides valuable means for modifying functionality and structural properties of different polymers. Tyrosinases catalyze the hydroxylation of various monophenols to the corresponding o-diphenols, and the subsequent oxidation of o-diphenols to the corresponding quinones, which are highly reactive and can further undergo non-enzymatic reactions to produce mixed melanins and heterogeneous polymers. Tyrosinases are also capable of oxidizing protein- and peptide-bound tyrosyl residues, resulting in the formation of inter- and intra-molecular crosslinks. Tyrosinases from apple (AT), potato (PT), the white rot fungus Pycnoporus sanguineus (PsT), the filamentous fungus Trichoderma reesei (TrT) and the edible mushroom Agaricus bisporus (AbT) were compared for their biochemical characteristics. The enzymes showed different features in terms of substrate specificity, stereo-specificity, inhibition, and ability to crosslink the model protein, -casein. All enzymes were found to produce identical semiquinone radicals from the substrates as analyzed by electron spin resonance spectroscopy. The result suggests similar reaction mechanism between the tyrosinases. PsT enzyme had the highest monophenolase/diphenolase ratio for the oxidation of monophenolic l-tyrosine and diphenolic l-dopa, although the tyrosinases generally had noticeably lower activity on monophenols than on di- or triphenols. The activity of AT and PT on tyrosine was particularly low, which largely explains the poor crosslinking ability of the model protein -casein by these enzymes. AbT oxidized peptide-bound tyrosine, but was not able to crosslink -casein. Conversely, the activity of PsT on model peptides was relatively low, although the enzyme could crosslink -casein. In the reaction conditions studied, TrT showed the best ability to crosslink -casein. TrT also had the highest activity on most of the tested monophenols, and showed noticeable short lag periods prior to the oxidation.     

Purification, characterization and molecular cloning of tyrosinase from the cephalopod mollusk, Illex argentinus.

Tyrosinase (monophenol, L-DOPA:oxygen oxidoreductase) was isolated from the ink of the squid, Illex argentinus. Squid tyrosinase, termed ST94, was found to occur as a covalently linked homodimeric protein with a molecular mass of 140.2 kDa containing two copper atoms per a subunit. The tyrosinase activity of ST94 was enhanced by proteolysis with trypsin to form a protein, termed ST94t, with a molecular mass of 127.6 kDa. The amino acid sequence of the subunit was deduced from N-terminal amino acid sequencing and cDNA cloning, indicating that the subunit of ST94 is synthesized as a premature protein with 625 amino acid residues and an 18-residue signal sequence region is eliminated to form the mature subunit comprised of 607 amino acid residues with a deduced molecular mass of 68,993 Da. ST94 was revealed to contain two putative copper-binding sites per a subunit, that showed sequence similarities with those of hemocyanins from mollusks, tyrosinases from microorganisms and vertebrates and the hypothetical tyrosinase-related protein of Caenorhabditis elegans. The squid tyrosinase was shown to catalyze the oxidation of monophenols as well as o-diphenols and to exhibit temperature-dependency of o-diphenolase activity like a psychrophilic enzyme.     

Kinetic study on the slow inhibition of epidermis tyrosinase by m-coumaric acid

The inhibition by m-coumaric acid of oxidation of L-dopa by epidermis tyrosinase (monophenol,dihydroxy-L-phenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is characterized by a prolonged transient phase. Kinetic data correspond to that for a postulated mechanism that involves rapid formation of a reduced enzyme-m-coumaric acid complex that subsequently undergoes a relatively slow reversible reaction. An overall inhibition constant for m-coumaric acid of 0.05 mM was calculated. The value of the Ki for the dissociation of m-coumaric acid from the rapidly formed complex was calculated as 0.53 mM. The first-order rate constants for the slow isomerization of the enzyme-inhibitor complex were calculated as 3.0 +/- 0.1 min-1 for the forward step and 0.31 +/- 0.06 min-1 for the reverse step.     

Extracellular tyrosinase from Streptomyces sp. KY-453: purification and some enzymatic properties

A strain of Streptomyces isolated from soil was found to produce a large amount of tyrosinase (monophenol, dihydroxy-L-phenylalanine: oxygen oxidoreductase: EC 1.14.18.1) extracellularly. The enzyme was purified from the culture filtrate about 550-fold by a series of column chromatographies on Duolite A-2 and CM-cellulose and gel filtration on Sephadex G-100. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the hydroxylation of monophenols and the oxidation of diphenols and was most active at pH 6.8 with dihydroxy-L-phenylalanine (L-DOPA) as the substrate. It was inhibited by kojic acid, diethyldithiocarbamate, and inhibitors obtained from micro-organisms. The isoelectric point of the enzyme was 9.9, and the molecular weight was estimated to be 36,000 by gel filtration on Sephadex G-100 and 29,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, which suggests that the enzyme is a monomer. Metal analysis by atomic absorption spectroscopy indicated that the enzyme contains nearly 1 gram atom of copper per mol.     

Properties of an enzyme from bananas (Musa sapientum) which hydroxylates tyramine to dopamine

An enzyme system isolated from the pulp of banana fruit (Musa sapientum) was partially purified and characterized. The enzyme was capable of catalysing the hydroxylation of the monophenol, tyramine, to the diphenol, dopamine (3,4-dihydroxyphenylethylamine). Unlike some tyrosinases, the reaction was not stimulated by catalytic amounts of diphenolic reaction product. Ascorbic acid, however, reduced the initial lag period in the oxidation of tyramine, stimulated the reaction rate and promoted the accumulation of dopamine during the first few minutes of the reaction. The hydroxylation of tyramine was apparently dependent upon molecular oxygen. On the basis of these observations it is tentatively suggested that the enzyme is a tyramine hydroxylase which may be responsible for the formation of dopamine in the banana.     

Purification, characterization, and molecular cloning of tyrosinase from Pholiota nameko

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.     

绞股蓝皂苷-硒配合物对酪氨酸酶的活性影响及动力学分析

为考察绞股蓝皂苷及其硒配合物对酪氨酸酶的动力学参数和作用机理。本研究采用体外酶促反应,以L-酪氨酸和L-DOPA为底物,模拟了酪氨酸酶单酚和二酚酶的体外催化氧化过程。绞股蓝总皂苷在50%、70%乙醇洗脱段和50%、70%乙醇洗脱绞股蓝皂苷-硒配合物在酪氨酸酶上的Ki值分别为1. 533、1. 767、1. 312和1. 210 mmol/L。Ki值越低,对酪氨酸酶的抑制作用越强,单酚酶的氧化阶段越快,表明硒元素显著提高了绞股蓝皂苷对酪氨酸酶的抑制作用。酶反应动力学分析表明,四种绞股蓝皂苷及其硒配合物对酪氨酸的抑制作用均为混合竞争抑制。其独特的药理化学特性为绞股蓝及硒系美白化妆品的进一步研究开发提供了理论依据和参考。     

Isolation and characterization of novel tyrosinase from Laceyella sacchari

We here describe the isolation and characterization of a tyrosinase from a newly isolated soil bacterium. 16S rDNA sequence analysis revealed that the bacterium most probably belongs to the species Laceyella sacchari (Ls) ( > 99.9 % identity). The tyrosinase extracellular enzymatic activity was induced in the presence of L-methionine and CuSO4. The crude enzyme was first purified by centrifugation followed by ammonium sulphate precipitation and ultrafiltration. After removal of a brown pigment, probably melanin, a purified enzyme was obtained by further separation of the crude protein mixture using size exclusion chromatography. Some 10.5 mg of pure tyrosinase (LsTyr) was isolated with a molecular mass of 30 910 Da, based on MALDI mass spectrometry. Together with the observed enzymatic activity, N-terminal chemical sequence analysis confirmed that the isolated enzyme is homologous to other tyrosinases. The kinetic parameters for the diphenol substrates L-DOPA and dopamine and for the monophenol substrate L-tyrosine were determined to be KM = 4.5 mM , 1.5 mM and 0.055 mM, and kcat/KM = 261.5 mM-1 s -1 , 30.6 mM-1 s-1 and 56.3 mM-1 s-1, respectively. Maximal activities of the purified enzyme were found to occur at pH 6.8.     

Indirect oxidation of 6-tetrahydrobiopterin by tyrosinase

6-Tetrahydrobiopterin is known to bind to an allosteric site of tyrosinase to directly inhibit the enzyme. However, simultaneous measurements of ultraviolet-visible absorption spectra and oxygen consumption led us to conclude that the inhibition was due to oxidation of 6-tetrahydrobiopterin by dopaquinone. Immediately after addition of 6-tetrahydrobiopterin, tyrosinase stopped producing dopachrome from either tyrosine or dopa. Duration of inhibition was proportional to the concentration of added 6-tetrahydrobiopterin and the enzyme activity was fully restored after the inhibition. Surprisingly, there was a rapid consumption of oxygen during the inhibition period. In addition, absorption spectra indicated that the only reaction that occurred during the inhibition was oxidation of 6-tetrahydrobiopterin to 7,8-dihydrobiopterin. In the absence of tyrosine or dopa, tyrosinase did not oxidize 6-tetrahydrobiopterin, suggesting that a reaction intermediate between dopa and dopachrome was a target for the inhibition. We propose a new mechanism in which dopa is oxidized to dopaquinone and the latter, instead of producing dopachrome, is reduced back to dopa by 6-tetrahydrobiopterin.     

New tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates

In this study, new tyrosinase inhibitors, (+)-catechin-aldehyde polycondensates, have been developed. Tyrosinase is a copper-containing enzyme that catalyzes the hydroxylation of a monophenol (monophenolase activity) and the oxidation of an o-diphenol (diphenolase activity). In the measurement of tyrosinase inhibition activity, (+)-catechin acted as substrate and cofactor of tyrosinase. On the other hand, the polycondensates inhibited the tyrosine hydroxylation and L-DOPA oxidation by chelation to the active site of tyrosinase. The UV-visible spectrum of a mixture of tyrosinase and the polycondensate exhibited a characteristic shoulder peak ascribed to the chelation of the polycondensate to the active site of tyrosinase. Furthermore, circular dichroism measurement showed a small red shift of the band due to the interaction between tyrosinase and the polycondensate. These data support that the polycondensate acts as an inhibitor of tyrosinase.     

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98×10(-6)-27.49×10(-6) M), good reproducibility, and high affinity to antioxidants (K(M) in the range of 62.31-144.87 μM). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds.     

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata)

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.     

Enzymatic synthesis of 3'-hydroxyacetaminophen catalyzed by tyrosinase

3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.     

Mechanistic implications of variable stoichiometries of oxygen consumption during tyrosinase catalyzed oxidation of monophenols and o-diphenols

The stoichiometry of oxygen consumption during tyrosinase-catalyzed oxidation of an o-diphenol (4-tert-butylcatechol, TBC) and a monophenol (4-tert-butylphenol, TBP) has been determined. At high [substrate]/[enzyme] ratios, in the case of o-diphenols, the stoichiometry of the enzyme-catalyzed reaction was always 1 O(2)/2 o-diphenols, although if the o-quinone product was unstable, the apparent stoichiometry could tend to 1 O(2)/1 o-diphenol due to regeneration of an o-diphenol in a side reaction. In the case of monophenols, the stoichiometry could be 1 O(2)/1 monophenol or 1.5 O(2)/1 monophenol depending if the o-quinone product was stable or unstable, respectively. However, at low [substrate]/[enzyme] ratios, the oxygen/substrate stoichiometry could, even in the case where stable products are formed, be lower than 1 O(2)/2 substrates for o-diphenols or higher than 1 O(2)/1 substrate for monophenols. These data supported the mechanism proposed by Rodríguez-López et al. [J. Biol. Chem. 267 (1992) 3801-3810], in which, during hydroxylation of monophenols, tyrosinase first transformed monophenol to o-diphenol and then either catalyzed a further oxidation to form o-quinone or released it into the reaction medium. In this second case, subsequent oxidation of the o-diphenol resulted in additional oxygen consumption.     

Enzymatic cross-linking of gelatine with laccase and tyrosinase

Conventional cross-linking of proteins involves the use of toxic chemicals. Here, cross-linking of gelatine and gelatine hydrolysates with tyrosinases from Botryosphaeria obtusa (BoT1 and BoT2), Agaricus bisporus (AbT) and from Verrucomicrobium spinosum (VsT) and with laccases from Trametes hirsuta (ThL) and T. versicolor (TvL) was demonstrated. Enzymatic oxidation of tyrosine residues was indicated by UV/VIS and fluorescence spectroscopy and further confirmed by oxygen consumption measurements. Using a model substrate (Tyr-Ala) dimerization was demonstrated by using RP-HPLC and LC-MS. Enzymatic cross-linking significantly increased the molecular weight of the soluble material up to the point of precipitation as demonstrated by both SDS-PAGE and size exclusion chromatography. The effect of cross-linking was further enhanced in the presence of phenolic molecules such as catechin.     

Selective inhibition of the oxidation of ferrous iron or sulfur in Thiobacillus ferrooxidans

The oxidation of either ferrous iron or sulfur by Thiobacillus ferrooxidans was selectively inhibited or controlled by various anions, inhibitors, and osmotic pressure. Iron oxidation was more sensitive than sulfur oxidation to inhibition by chloride, phosphate, and nitrate at low concentrations (below 0.1 M) and also to inhibition by azide and cyanide. Sulfur oxidation was more sensitive than iron oxidation to the inhibitory effect of high osmotic pressure. These differences were evident not only between iron oxidation by iron-grown cells and sulfur oxidation by sulfur-grown cells but also between the iron and sulfur oxidation activities of the same iron-grown cells. Growth experiments with ferrous iron or sulfur as an oxidizable substrate confirmed the higher sensitivity of iron oxidation to inhibition by phosphate, chloride, azide, and cyanide. Sulfur oxidation was actually stimulated by 50 mM phosphate or chloride. Leaching of Fe and Zn from pyrite (FeS(2)) and sphalerite (ZnS) by T. ferrooxidans was differentially affected by phosphate and chloride, which inhibited the solubilization of Fe without significantly affecting the solubilization of Zn.     

Identification of oxidation product of arbutin in mushroom tyrosinase assay system

In order to elucidate whitening mechanisms of arbutin (hydroquinone-O-beta-D-glucopyranoside), its effects on mushroom tyrosinase were analyzed by spectrophotometric, polarographic, and HPLC experiments. It was found that as soon as catalytic amounts of L-DOPA become available as a cofactor, arbutin acts as a monophenol substrate. A significant enzymatic product was identified as 3,4-dihydroxyphenyl-O-beta-D-glucopyranoside by NMR and MS experiments.     

Inhibition of the catecholase activity of biomimetic dinuclear copper complexes by kojic acid

The inhibition of the catechol oxidase activity exhibited by three dinuclear copper(II) complexes, derived from different diaminotetrabenzimidazole ligands, by kojic acid [5-hydroxy-2-(hydroxymethyl)-γ-pyrone] has been studied. The catalytic mechanism of the catecholase reaction proceeds in two steps and for both of these inhibition by kojic acid is of competitive type. The inhibitor binds strongly to the dicopper(II) complex in the first step and to the dicopper-dioxygen adduct in the second step, preventing in both cases the binding of the catechol substrate. Binding studies of kojic acid to the dinuclear copper(II) complexes and a series of mononuclear analogs, carried out spectrophotometrically and by NMR, enable us to propose that the inhibitor acts as a bridging ligand between the metal centers in the dicopper(II) catalysts. Received: 23 August 1999 / Accepted: 20 January 2000