Fluorescence detection of valence speciation of Cr(III) based on the catechol oxidase mimic enzyme activity of CuSeNP nanozymes.

Copper selenide nanoparticles (CuSeNP) were synthesized using histidine, ethylenediamine, and sodium selenate as precursors by one-step microwave digestion methods. The as-prepared CuSeNPs exhibit excellent catechol oxidase mimic enzyme and catalase (CAT)-like activities. Dopamine (DA) can be oxidized to aminochrome with H2O2 by CuSeNPs, and the intermediate product aminochrome can further react with α-naphthol to yield a highly fluorescent derivative. It was confirmed that Cr(III) could adsorb on the surface of CuSeNPs and inhibit the production of semiquinone radicals in the reaction system, and the catalytic activity of CuSeNPs was inhibited. The detection mechanisms, kinetics, and catalytic properties of CuSeNPs were systematically investigated. As a result, a novel fluorescence method for the assay of Cr(III) was established. The feasibility of CuSeNP nanozyme in detecting speciation Cr(III) in food samples was explored with satisfactory results. It showed the obvious potential for developing effective and dependable fluorescent detection method for protecting food safety.

Amino-Ligand-Coordinated Dicopper Active Sites Enable Catechol Oxidase-Like Activity for Chiral Recognition and Catalysis.

Developing highly active and selective advanced nanozymes for enzyme-mimicking catalysis remains a long-standing challenge for basic research and practical applications. Herein, we grafted a chiral histidine- (His-) coordinated copper core onto Zr-based metal-organic framework (MOF) basic backbones to structurally mirror the bimetal active site of natural catechol oxidase. Such a biomimetic fabricated process affords MOF-His-Cu with catechol oxidase-like activity, which can catalyze dehydrogenation and oxidation of o-diphenols and then transfer electrons to O2 to generate H2O2 by the cyclic conversion of Cu(II) and Cu(I). Specifically, the elaborate incorporation of chiral His arms results in higher catalytic selectivity over the chiral catechol substrates than natural enzyme. Density functional theory calculations reveal that the binding energy and potential steric effect in active site-substrate interactions account for the high stereoselectivity. This work demonstrates efficient and selective enzyme-mimicking catalytic processes and deepens the understanding of the catalytic mechanism of nanozymes.

Substrate specificity of polyphenol oxidase.

The ubiquitous type-3 copper enzyme polyphenol oxidase (PPO) has found itself the subject of profound inhibitor research due to its role in fruit and vegetable browning and mammalian pigmentation. The enzyme itself has also been applied in the fields of bioremediation, biocatalysis and biosensing. However, the nature of PPO substrate specificity has remained elusive despite years of study. Numerous theories have been proposed to account for the difference in tyrosinase and catechol oxidase activity. The "blocker residue" theory suggests that bulky residues near the active site cover CuA, preventing monophenol coordination. The "second shell" theory suggests that residues distant (∼8 Å) from the active site, guide and position substrates within the active site based on their properties e.g., hydrophobic, electrostatic. It is also hypothesized that binding specificity is related to oxidation mechanisms of the catalytic cycle, conferred by coordination of a conserved water molecule by other conserved residues. In this review, we highlight recent developments in the structural and mechanistic studies of PPOs and consolidate key concepts in our understanding toward the substrate specificity of PPOs.

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases.

Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze theo-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their--so far unknown--natural substrates in vivo.

Tyrosinase and catechol oxidase activity of copper(I) complexes supported by imidazole-based ligands: structure-reactivity correlations.

Four new imidazole-based ligands, 4-((1H-imidazol-4-yl)methyl)-2-phenyl-4,5-dihydrooxyzole (L OL 1), 4-((1H-imidazol-4-yl)methyl)-2-(tert-butyl)-4,5-dihydrooxyzole (L OL 2), 4-((1H-imidazol-4-yl)methyl)-2-methyl-4,5-dihydrooxyzole (L OL 3), and N-(2,2-dimethylpropylidene)-2-(1-trityl-1H-imidazol-4-yl-)ethyl amine (L imz 1), have been synthesized. The corresponding copper(I) complexes [Cu(I)(L OL 1)(CH3CN)]PF6 (CuL OL 1), [Cu(I)(L OL 2)(CH3CN)]PF6 (CuL OL 2), [Cu(I)(L OL 3)(CH3CN)]PF6 (CuL OL 3), [Cu(I)(L imz 1)(CH3CN)2]PF6 (CuL imz 1) as well as the Cu(I) complex derived from the known ligand bis(1-methylimidazol-2-yl)methane (BIMZ), [Cu(I)(BIMZ)(CH3CN)]PF6 (CuBIMZ), are screened as catalysts for the oxidation of 3,5-di-tert-butylcatechol (3,5-DTBC-H2) to 3,5-di-tert-butylquinone (3,5-DTBQ). The primary reaction product of these oxidations is 3,5-di-tert-butylsemiquinone (3,5-DTBSQ) which slowly converts to 3,5-DTBQ. Saturation kinetic studies reveal a trend of catalytic activity in the order CuL OL 3 ≈ CuL OL 1 > CuBIMZ > CuL OL 2 > CuL imz 1. Additionally, the catalytic activity of the copper(I) complexes towards the oxygenation of monophenols is investigated. As substrates 2,4-di-tert-butylphenol (2,4-DTBP-H), 3-tert-butylphenol (3-TBP-H), 4-methoxyphenol (4-MeOP-H), N-acetyl-L-tyrosine ethyl ester monohydrate (NATEE) and 8-hydroxyquinoline are employed. The oxygenation products are identified and characterized with the help of UV/Vis and NMR spectroscopy, mass spectrometry, and fluorescence measurements. Whereas the copper complexes with ligands containing combinations of imidazole and imine functions or two imidazole units (CuL imz 1 and CuBIMZ) are found to exhibit catalytic tyrosinase activity, the systems with ligands containing oxazoline just mediate a stoichiometric conversion. Correlations between the structures of the complexes and their reactivities are discussed.

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.

Selective sensing of catechol based on a fluorescent nanozyme with catechol oxidase activity.

Nanozymes, an unusual category of nanomaterials possessing enzymatic properties, and have generated considerable interest regarding their application feasibilities on several important fronts. In the present work, an innovative sensing device for catechol was established ground on a fluorescent nanozyme (Cu-BDC-NH2) that exhibited catechol oxidase activity. The fluorescent nanozyme combines both functions of catechol recognition and response signal output, and can realize the sensing of catechol without the addition of other chromogenic agents. In the existence of Cu-BDC-NH2, catechol can be oxidized efficiently to produce quinones or polymers with strong electron absorption capacity, which immediately results in efficient fluorescence quenching of Cu-BDC-NH2. However, other common phenolic compounds, such as phenol, the other two diphenols (hydroquinone and resorcinol), phloroglucinol, and chlorophenol, do not result in efficient fluorescence quenching of Cu-BDC-NH2. The method shows a nice linear relationship between catechol concentration prep the fluorescence intensity of Cu-BDC-NH2 in the scope of 0-10 µM, with a detection limit of 0.997 µM. The detection of catechol in actual water samples has also achieved the satisfactory consequences, which provides a new strategy for the convenient and selective detection of catechol.

Catechol oxidase nanozyme based colorimetric sensors array for highly selective distinction among multiple catecholamines.

In order to effectively monitor multiple catecholamine (CA) neurotransmitters with extreme similar structures, a rapid, sensitive and selective detection strategy has become an urgent problem to be solved. In this paper, a novel colorimetric sensors array based on CuNCs protected by various ligands such as tannic acid, ascorbic acid and polymethylacrylic acid (CuNCs@TA, CuNCs@AA and CuNCs@PMAA) was constructed. All of these CuNCs could mimic catechol oxidase to selective catalyze catechol-type analogues (such as CAs) to corresponding quinones along with color changes. Furthermore, experiments and theory calculations demonstrated that Cr6+-modification on the surface of CuNCs facilitated the steady-state kinetics of enzymatic activity. Based on these CuNCs as sensing probes, this sensors array can quickly detect different CAs (such as epinephrine (EP), including dopamine (DA), norepinephrine (NE) and l-dopa) with similar structures. When those analogues were added to the CuNC-based colorimetric array sensors, different absorbance changes were produced at 485 nm. Linear discriminant analysis (LDA) showed that the tri-probe colorimetric array sensors could recognize and distinguish these analogues, and corresponding binary and ternary mixtures could be well categorized. The value of Factor 1 of an array with varied CA concentrations had a good linear correlation, and the detection limit (LOD) was as low as 10-8∼10-9 mol/L. Four CA analogues in real samples were identified by CuNCs-based colorimetric array sensors. This work provides a fast and convenient experimental basis for monitoring the complex structure CAs neurotransmitters.

New mechanistic insights into coupled binuclear copper monooxygenases from the recent elucidation of the ternary intermediate of tyrosinase.

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2 /monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the µ-η2 :η2 -peroxide dicopper(II) O-O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non-protonated µ-oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure-function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites.

Purification and characterization of catechol oxidase from Tadela (Phoenix dactylifera L.) date fruit.

Catechol oxidase (PPO) was extracted and purified from Tadela (Phoenix dactylifera L.) date fruit, by a procedure that included (NH4)2SO4 precipitation followed by dialysis, Q-Sepharose bb ion-exchange chromatography and HPLC gel filtration chromatography. Some of its biochemical characteristics were studied. The purification rate and the yield were 80% and 20%, respectively. The Tadela date fruit catechol oxidase exhibited a molecular weight of 90 kDa using SDS-PAGE. The catechol oxidase showed only o­diphenolase and triphenolase activities while no monophenolase activity was detected. A better affinity was observed using catechol as substrate (Km = 35 mM) with thus, a higher Vmax/Km ratio (80 U/mM·mL). This enzyme is thermostable in the temperature range (30-60 °C) with optimum activity in acidic range of pH. Four inhibitors were used for the control of enzymatic browning, of which sodium metabisulfite was the most potent (IC50 = 0, 11 mM). The values of KI and mechanism of inhibition were also determined. No significant change on enzyme activity was noticed in the presence of metal ion and detergents. Therefore, thermal inactivation was studied in the temperature range between 60 and 80 °C using catechol as substrate. Their kinetic (K, D, t1/2, Zt, Ea) and thermodynamic (ΔH, ΔG and ΔS) parameters were also estimated.

Emergence of hydrogen bonds from molecular dynamics simulation of substituted N-phenylthiourea-catechol oxidase complex.

A series of N-phenylthiourea derivatives was built starting from the X-ray structure in the molecular mechanics framework and the interaction profile in the complex with the catechol oxidase was traced using molecular dynamics simulation. The results showed that the geometry and interactions between ligand and receptor were highly related to the position of the substituted side chains of phenyl moiety. At the end of molecular dynamics run, a concentrated multicenter hydrogen bond was created between the substituted ligand and receptor. The conformation of the ligand itself were also restricted in the receptor pocket. Furthermore, the simulation time of 50 ns were found to be long enough to explore the relevant conformational space and the stationary behavior of the molecular dynamic could be observed.

Boosting LPMO-driven lignocellulose degradation by polyphenol oxidase-activated lignin building blocks.

BACKGROUND: Many fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). The application of LPMOs is expected to contribute to ecologically friendly conversion of biomass into fuels and chemicals. Moreover, applications of LPMO-modified cellulose-based products may be envisaged within the food or material industry. RESULTS: Here, we show an up to 75-fold improvement in LPMO-driven cellulose degradation using polyphenol oxidase-activated lignin building blocks. This concerted enzymatic process involves the initial conversion of monophenols into diphenols by the polyphenol oxidase MtPPO7 from Myceliophthora thermophila C1 and the subsequent oxidation of cellulose by MtLPMO9B. Interestingly, MtPPO7 shows preference towards lignin-derived methoxylated monophenols. Sequence analysis of genomes of 336 Ascomycota and 208 Basidiomycota reveals a high correlation between MtPPO7 and AA9 LPMO genes. CONCLUSIONS: The activity towards methoxylated phenolic compounds distinguishes MtPPO7 from well-known PPOs, such as tyrosinases, and ensures that MtPPO7 is an excellent redox partner of LPMOs. The correlation between MtPPO7 and AA9 LPMO genes is indicative for the importance of the coupled action of different monooxygenases in the concerted degradation of lignocellulosic biomass. These results will contribute to a better understanding in both lignin deconstruction and enzymatic lignocellulose oxidation and potentially improve the exploration of eco-friendly routes for biomass utilization in a circular economy.

Platinum Nanoparticles: Efficient and Stable Catechol Oxidase Mimetics.

Although enzyme-like nanomaterials have been extensively investigated over the past decade, most research has focused on the peroxidase-like, catalase-like, or SOD-like activity of these nanomaterials. Identifying nanomaterials having oxidase-like activities has received less attention. In this study, we demonstrate that platinum nanoparticles (Pt NPs) exhibit catechol oxidase-like activity, oxidizing polyphenols into the corresponding o-quinones. Four unique approaches are employed to demonstrate the catechol oxidase-like activity exerted by Pt NPs. First, UV-vis spectroscopy is used to monitor the oxidation of polyphenols catalyzed by Pt NPs. Second, the oxidized products of polyphenols are identified by ultrahigh-performance liquid chromatography (UHPLC) separation followed by high-resolution mass spectrometry (HRMS) identification. Third, electron spin resonance (ESR) oximetry techniques are used to confirm the O2 consumption during the oxidation reaction. Fourth, the intermediate products of semiquinone radicals formed during the oxidation of polyphenols are determined by ESR using spin stabilization. These results indicate Pt NPs possess catechol oxidase-like activity. Because polyphenols and related bioactive substances have been explored as potent antioxidants that could be useful for the prevention of cancer and cardiovascular diseases, and Pt NPs have been widely used in the chemical industry and medical science, it is essential to understand the potential effects of Pt NPs for altering or influencing the antioxidant activity of polyphenols.

Structure-function correlations in tyrosinases.

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure-function correlations in tyrosinases along with comparison to other type-3 copper proteins.

Purification and characterization of tyrosinase from walnut leaves (Juglans regia).

Polyphenol oxidase (PPO) is a type-3 copper enzyme catalyzing the oxidation of phenolic compounds to their quinone derivates, which are further converted to melanin, a ubiquitous pigment in living organisms. In this study a plant originated tyrosinase was isolated from walnut leaves (Juglans regia) and biochemically characterized. It was possible to isolate and purify the enzyme by means of an aqueous two-phase extraction method followed by chromatographic purification and identification. Interestingly, the enzyme showed a rather high monophenolase activity considering that the main part of plant PPOs with some exceptions solely possess diphenolase activity. The average molecular mass of 39,047 Da (Asp(101)→Arg(445)) was determined very accurately by high resolution mass spectrometry. This proteolytically activated tyrosinase species was identified as a polyphenol oxidase corresponding to the known jrPPO1 sequence by peptide sequencing applying nanoUHPLC-ESI-MS/MS. The polypeptide backbone with sequence coverage of 96% was determined to start from Asp(101) and not to exceed Arg(445).

Type-3 copper proteins: recent advances on polyphenol oxidases.

Recent investigations in the study of plant, fungal, and bacterial type-3 copper proteins are reviewed. Focus is given to three enzymes: catechol oxidases (CO), tyrosinases, and aureusidin synthase. CO were mostly found in plants, however, in 2010 the first fungal CO was published. The first plant-originated tyrosinase was published in 2014, before tyrosinases were only reported in fungi, bacteria, and human. Aureusidin synthase from yellow snapdragon (Antirrhinum majus) was first published in 2000, as part of yellow flower coloration pathway. In the last years, many important results on type-3 copper enzymes originated from X-ray crystallographic investigations. In addition, studies on site-directed mutagenesis of amino acids around the active site were performed to identify the regions determining monophenolase and/or diphenolase activity. Although X-ray crystallographic structures of CO and tyrosinases are available, many questions like the response for the activation via proteases, sequence-based or structural-based differences between CO, as well as the physiological roles of many polyphenol oxidases still remain to be addressed.