Catechol Oxidase versus Tyrosinase Classification Revisited by Site‐Directed Mutagenesis Studies

Catechol oxidases (COs) and tyrosinases (TYRs) are both polyphenol oxidases (PPOs) that catalyze the oxidation of ortho‐diphenols to the corresponding quinones. By the official classification, only TYRs can also catalyze the hydroxylation of monophenols to ortho‐diphenols. Researchers have been trying to find the molecular reason for the mono‐/diphenolase specificity for decades. However, the hypotheses for the lack of monophenolase activity of plant COs are only based on crystal structures so far. To test these hypotheses, we performed site‐directed mutagenesis studies and phylogenetic analyses with dandelion PPOs offering high phylogenetic diversity, the results of which refute the structure‐based hypotheses. While plant PPOs of phylogenetic group 2 solely exhibit diphenolase activity, plant PPOs of phylogenetic group 1 unexpectedly also show monophenolase activity. This finding sheds new light upon the molecular basis for mono‐/diphenol substrate specificity and challenges the current practice of generally naming plant PPOs as COs.     

Enzyme Modification and Oxidative Cross‐linking Using Carboxylate‐, Phenol‐ and Catechol‐Conjugated 1,8‐Naphthalimides

The ground‐ and excited‐state interactions of β‐alanine, tyrosine and l ‐dopa substituted 1,8 naphthalimides (NI‐Ala, NI‐Tyr and NI‐Dopa) with lysozyme and mushroom tyrosinase were evaluated to understand the mechanism of oxidative modification. Photooxidative cross‐linking of lysozyme was observed for all three conjugates. The yield was significantly reduced for NI‐Tyr and NI‐Dopa due to intramolecular electron transfer to the excited singlet state of the 1,8‐naphthalimide. Incubation of NI‐Tyr and NI‐Dopa with mushroom tyrosinase resulted in an increased fluorescence from the naphthalimide, suggesting that the phenol and catechol portion of the conjugates are oxidized by the enzyme. This result demonstrates that the compounds bind in the active site of mushroom tyrosinase. The catalytic activity of mushroom tyrosinase to oxidize both tyrosine (monophenolase) and l ‐dopa (diphenolase) was modified by NI‐Tyr and NI‐Dopa. Monophenolase activity was inhibited, and the diphenolase activity was enhanced in the presence of these conjugates. Detailed Michaelis–Menten studies show that both V_max and K_m are modified, consistent with a mixed inhibition mechanism. Collectively, the results show that the compounds interact in the enzyme's active site, but also modify the distribution of the enzyme's oxidation states that are responsible for catalysis.     

含三氟甲基1,2,3-三氮唑衍生物对蘑菇酪氨酸酶活性的抑制动力学(英文)

研究表明,含三氟甲基1,2,3-三氮唑衍生物(TF-TZ)对蘑菇酪氨酸酶具有很强的抑制性:不仅抑制单酚酶活性,而且对二酚酶活性也是可逆性抑制,其半抑制浓度IC50分别为30.4和34.5μmol.L-1,并且单酚酶延滞时间随着TF-TZ浓度增加而延长.TF-TZ对二酚酶的抑制为抛物线型竞争性抑制,表明一分子的酶可以和两分子的TF-TZ相结合,从而形成两种酶-抑制剂复合物:EI和EI2,其抑制常数分别为76.9和9.71μmol.L-1.紫外-可见光谱表明,TF-TZ和酶相互作用后产成特征的"肩峰",说明TF-TZ能够作用于酶的活性中心.     

Tyrosinase versus Catechol Oxidase: One Asparagine Makes the Difference

Tyrosinases mediate the ortho‐hydroxylation and two‐electron oxidation of monophenols to ortho‐quinones. Catechol oxidases only catalyze the oxidation of diphenols. Although it is of significant interest, the origin of the functional discrimination between tyrosinases and catechol oxidases has been unclear. Recently, it has been postulated that a glutamate and an asparagine bind and activate a conserved water molecule towards deprotonation of monophenols. Here we demonstrate for the first time that a polyphenoloxidase, which exhibits only diphenolase activity, can be transformed to a tyrosinase by mutation to introduce an asparagine. The asparagine and a conserved glutamate are necessary to properly orient the conserved water in order to abstract a proton from the monophenol. These results provide direct evidence for the crucial importance of a proton shuttle for tyrosinase activity of type 3 copper proteins, allowing a consistent understanding of their different chemical reactivities.     

A Novel Heptapeptide with Tyrosinase Inhibitory Activity Identified from a Phage Display Library

Peptidic inhibition of the enzyme tyrosinase, responsible for skin pigmentation and food browning, would be extremely useful for the food, cosmetics, and pharmaceutical industries. In order to identify novel inhibitory peptides, a library of short sequence oligopeptides was screened to reveal direct interaction with the tyrosinase. A phage displaying heptapeptide (IQSPHFF) was found to bind most strongly to tyrosinase. The inhibitory activity of the heptapeptide was evaluated using mushroom tyrosinase. The results showed that the peptide inhibited both the monophenolase and diphenolase activities of mushroom tyrosinase with IC₅₀ values of 1.7 and 4.0 mM, respectively. The heptapeptide is thought to be a reversible competitive inhibitor of diphenolase with the inhibition constants (Ki) of 0.765 mM. To further investigate how the heptapeptide exerts its inhibitory effect, a docking study between tyrosinase and heptapeptide was performed. The simulation showed that the heptapeptide binds in the active site of the enzyme near the catalytically active Cu ions and forms hydrogen bonds with five histidine residues on the active site. Phage display technology is thus a useful approach for the screening of potential tyrosinase inhibitors and could be widely applicable to a much wider range of enzymes.     

Inhibition Kinetics of Cabbage Butterfly (<Emphasis Type="Italic">Pieris rapae</Emphasis> L.) Larvae Phenoloxidase Activity by 3-Hydroxy-4-Methoxybenzaldehyde Thiosemicarbazone

Phenoloxidase (PO) is a key enzyme in insect development, responsible for catalyzing the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the present study, the kinetic assay in air-saturated solutions and the kinetic behavior of PO from Pieris rapae (Lepidoptera) larvae in the oxidation of l-tyrosine (a monophenol) and l-DOPA (l-3, 4-dihydroxyphenylalanine) (a diphenol) was studied. The inhibitory effects of 3-hydroxy-4-methoxybenzaldehyde thiosemicarbazone (3-H-4-MBT) on the monophenolase and diphenolase activities of PO were also studied. The results show that 3-H-4-MBT can inhibit both the monophenolase and diphenolase activities of PO. The lag period of l-tyrosine oxidation catalyzed by the enzyme was obviously lengthened and the steady-state activities of the enzyme sharply decreased. The inhibitor was found to be noncompetitively reversible with a K _I (K _I = K _IS) of 0.30 μmol/L and an estimated IC₅₀ of 0.14 ± 0.02 μmol/L for monophenolase and 0.26 ± 0.04 μmol/L for diphenolase. In the time course of the oxidation of l-DOPA catalyzed by the enzyme in the presence of different concentrations of 3-H-4-MBT, the rate decreased with increasing time until a straight line was approached. The microscopic rate constants for the reaction of 3-H-4-MBT with the enzyme were determined.     

Inhibitory Effects of Fatty Acids on the Activity of Mushroom Tyrosinase

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.     

Tyrosinase inhibition kinetic studies of standardized extract of Berberis aristata

The stem bark and wood of Berberis aristata DC (Daruharidra) are one of the principal ingredients of traditional skin lighting and exfoliating scrub preparation in India. The standardised extract of B. aristata was screened to evaluate their in vitro antityrosinase activity and inhibition kinetics. Phytochemical and pharmacological studies were carried out with different solvent fractions of the methanol extract of B. aristata (MEBA). RP-HPLC analysis was used to determine the berberine content in extract and fractions of B. aristata. MEBA showed maximum berberine content. Extract and fractions of B. aristata contain the maximum amount of alkaloids than other constituents. In tyrosinase inhibition assay, MEBA was found to possess highest dose-dependent monophenolase and moderate diphenolase activity. The enzyme kinetic study revealed that MEBA possessed mixed type inhibition of monophenolase activity of tyrosinase. These bioactivities indicate that the MEBA has antihyperpigmentation potential in human skin.     

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at −0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.     

Bromination of Cycloparaphenylenes: Strain‐Induced Site‐Selective Bis‐Addition and Its Application for Late‐Stage Functionalization

Bromination of [n ]cycloparaphenylenes (CPPs) is herein reported. Small [n ]CPPs (n <8) underwent a bis‐bromine addition reaction with high site selectively to produce tetrabromo adducts in moderate to excellent yields. Theoretical calculations revealed that thermodynamic stability dictates both the reactivity and site selectivity of the reaction. The addition product was further converted into the octabromo product by a FeBr₃‐catalyzed site‐selective bromination reaction. The tetra‐ and octabromine adducts were then transformed into mono‐ to tetrabromo CPPs, which were further converted into several CPP derivatives. Therefore, bromination and subsequent transformations provide a path for late‐stage functionalization of CPPs.     

A Highly Efficient Synthesis of Optically Active Ferrocenylethylamines via Hydride Reduction of Chiral Ferrocenylketimines

Due to using (R)‐ or (S)‐α‐methylbenzylamine as a chiral auxiliary, and low‐temperature regime for reduction of the intermediate ferrocenyl‐mono‐ or 1,1′‐bis‐ketimines, the corresponding secondary mono‐ or 1,1′‐bis‐amines were prepared with high diastereoselectivity. Removal of the α‐methylbenzyl group afforded the optically active primary mono‐ and bis‐ferrocenylethylamines in high yields. The absolute configuration of (R,R)‐ 3a and (S,S)‐ 3b was determined by X‐ray single crystal diffraction.     

The More Gold—The More Enantioselective: Cyclohydroaminations of γ‐Allenyl Sulfonamides with Mono‐, Bis‐, and Trisphospholane Gold(I) Catalysts

A series of chiral mono‐, di‐, and trinuclear gold(I) complexes have been prepared and used as precatalysts in the asymmetric cyclohydroamination of N‐protected γ‐allenyl sulfonamides. The stereodirecting ligands were mono‐, di‐, and tridentate 2,5‐diphenylphospholanes, which possessed C₁, C₂, and C₃ symmetry, respectively, thereby rendering the catalytic sites in the di‐ and trinuclear complexes symmetry equivalent. The C₃‐symmetric trinuclear complex displayed the highest activity and enantioselectivity (up to 95 % ee), whilst its mono‐ and dinuclear counterparts exhibited considerably lower enantioselectivities and activities. A similar trend was observed in a series of mono‐, di‐, and trinuclear 2,5‐dimethylphospholane gold(I) complexes. Aurophilic interactions were established from the solid‐state structures of the trinuclear gold(I) complexes, thereby raising the question as to whether these secondary forces were responsible for the different catalytic behavior observed.     

Effects of Active‐Site Modification and Quaternary Structure on the Regioselectivity of Catechol‐O‐Methyltransferase

Catechol‐O‐methyltransferase (COMT), an important therapeutic target in the treatment of Parkinson's disease, is also being developed for biocatalytic processes, including vanillin production, although lack of regioselectivity has precluded its more widespread application. By using structural and mechanistic information, regiocomplementary COMT variants were engineered that deliver either meta‐ or para‐methylated catechols. X‐ray crystallography further revealed how the active‐site residues and quaternary structure govern regioselectivity. Finally, analogues of AdoMet are accepted by the regiocomplementary COMT mutants and can be used to prepare alkylated catechols, including ethyl vanillin.     

Deprotonation‐Induced Aromaticity Enhancement and New Conjugated Networks in meso‐Hexakis(pentafluorophenyl)[26]hexaphyrin

meso‐Hexakis(pentafluorophenyl)‐substituted neutral hexaphyrin with a 26π‐electronic circuit can be regarded as a real homolog of porphyrin with an 18π‐electronic circuit with respect to a quite flat molecular structure and strong aromaticity. We have investigated additional aromaticity enhancement of meso‐hexakis(pentafluorophenyl)[26]hexaphyrin(1.1.1.1.1.1) by deprotonation of the inner N? H groups in the macrocyclic molecular cavity to try to induce further structural planarization. Deprotonated mono‐ and dianions of [26]hexaphyrin display sharp B‐like bands, remarkably strong fluorescence, and long‐lived singlet and triplet excited‐states, which indicate enhanced aromaticity. Structural, spectroscopic, and computational studies have revealed that deprotonation induces structural deformations, which lead to a change in the main conjugated π‐electronic circuit and cause enhanced aromaticity.     

From Packed “Sandwich” to “Russian Doll”: Assembly by Charge‐Transfer Interactions in Cucurbit[10]uril

As the host possessing the largest cavity in the cucurbit[n]uril (CB[n]) family, CB[10] has previously displayed unusual recognition and assembly properties with guests but much remains to be explored. Herein, we present the recognition properties of CB[10] toward a series of bipyridinium guests including the tetracationic cyclophane known as blue box along with electron‐rich guests and detail the influence of encapsulation on the charge‐transfer interactions between guests. For the mono‐bipyridinium guest (methylviologen, MV ²⁺), CB[10] not only forms 1:1 and 1:2 inclusion complexes, but also enhances the charge‐transfer interactions between methylviologen and dihydroxynaphthalene ( HN ) by mainly forming the 1:2:1 packed “sandwich” complex (CB[10] ? 2 MV ²⁺ ?HN ). For guest 1 with two bipyridinium units, an interesting conformational switching from linear to “U” shape is observed by adding catechol to the solution of CB[10] and the guest. For the tetracationic cyclophane‐blue box, CB[10] forms a stable 1:1 inclusion complex; the two bipyridinium units tilt inside the cavity of CB[10] according to the X‐ray crystal structure. Finally, a supramolecular “Russian doll” was built up by threading a guest through the cavities of both blue box and CB[10].     

羧基取代类立方烷结构的萘光二聚体与芳香胺的反应性

本文利用羧基取代类立方烷结构的萘光二聚体2与芳香胺3反应研究这类结构化合物的反应活性。X-单晶衍射分析表明二环已基碳二亚胺（DCC）与2生成了具有类立方烷骨架的稳定化合物4,而且DCC能够作为一种有效的缩合剂催化2与3的反应,得到单独用光化学方法或热化学方法都无法获得的单边缩合或双边缩合的类立方烷酰胺化合物5和6。研究发现芳香胺3的电子效应显著影响反应产物的分布。     

聚醚桥连二异羟肟酸单核和双核过渡金属配合物催化PNPP水解

Two polyether bridged dihydroxamic acids and their mono-and binuclear manganese(Ⅱ), zinc(Ⅱ) complexes have been synthesized and employed as models to mimic hydrolase in catalytic hydrolysis of p-nitrophenyl picolinate (PNPP). The reaction kinetics and the mechanism of hydrolysis of PNPP have been investigated. The kinetic mathematical model for PNPP cleaved by the complexes has been proposed. The effects of the different central metal ion, mono-and binuclear metal, the pseudo-macrocyclic polyether constructed by polyethoxy group of the complexes, and reactive temperature on the rate for catalytic hydrolysis of PNPP have been examined. The results showed that the transition metal dthydroxamates exhibited high catalytic activity to the hydrolysis of PNPP, the catalytic activity of binuclear complexes was higher than that of mononuclear ones, and the pseudo-macrocyclic polyether might synergetically activate H20 coordinated to metal ion with central metal ion together and promote the catalytic hydrolysis of PNPP.     

Solvent‐ and Catalyst‐Free Selective Mannich Reaction on Catechols and Para Substituted Phenols: A Convenient Route to Catechol‐ and Phenol‐Iminodiacetic Acid Ligands

Abstract

We have developed a convenient solvent‐ and catalyst‐free selective Mannich reaction of a variety of para‐substituted phenols or catechols with paraformaldehyde and ethyl iminodiacetate. With para‐substituted phenols and electron‐poor catechol, only the monosubstituted benzyliminodiacetic ester is selectively formed in good yield and no disubstituted product is detected. In contrast, electron rich catechols gave mono‐ or disubstituted derivatives depending on the stoichiometry of ethyliminodiacetate. Furthermore, the reaction is highly regioselective with catechols, since no para derivatives are formed. In addition, we describe a mild acidic hydrolysis of the ester functions, which avoids the degradation of the benzylamine moiety by the quinone methide pathway. So, pure o‐hydroxybenzyliminodiacetic acid ligands are obtained in overall good yields.     

Characterization of the Fe(III)‐binding Site in Sepia Eumelanin by Resonance Raman Confocal Microspectroscopy¶

The resonance Raman spectrum of Sepia eumelanin is discussed by analogy to model compounds containing catechol (CAT)‐like structural units. These data are then compared with the analogous data on Fe(III)‐enriched Sepia eumelanin. In contrast to the natural eumelanin, the Fe(III)‐enriched samples exhibit absorption features in the visible and near‐IR spectral regions, which are attributed to ligand‐to‐metal charge‐transfer (LMCT) bands. Resonance Raman spectra collected by exciting these LMCT bands reveal bands at 580 and 1470 cm^?1; the intensity of these features increases wioth increasing Fe(III) content. The 580 and 1470 cm^?1 bands are assigned to Fe‐OR stretching and ring deformation modes, respectively. These data further substantiate that the Fe(III)‐ melanin‐binding site in melanin is composed of CAT‐like structural units.     

Immobilisation of tyrosinase on siliceous cellular foams affording highly effective and stable biocatalysts

Tyrosinase from Agaricus bisporus was immobilised covalently on mesostructured siliceous foam (MCF) and three mesoporous silicas of SBA-15 type of different pore sizes, regarded as the reference, to reveal that MCF was the superior enzyme support. All the carriers were functionalised using 3-aminopropyltrimethoxysilane and the enzyme was attached covalently via glutaraldehyde or by simple adsorption and it was also cross-linked with glutaraldehyde in selected samples. The experiments indicated that only tyrosinase attached covalently was highly active and that postimmobilisation cross-linking slightly reduced its activity with no improvement in stability. MCF-bound tyrosinase was the best biocatalyst with monophenolase and diphenolase activities of 3627 U mL^?1 and 53040 U mL^?1 of carrier sediment, respectively. Inactivation studies at 55°C showed that MCF-bound tyrosinase was 20 times more stable than the native enzyme, whereas for typical SBA-15 it was only 12 times. A comparative study with other, non-siliceous enzyme supports indicated that aminated MCF appeared to be the carrier of choice for the covalent attachment of tyrosinase.