Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources.

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 µM), mushroom (Ki: 0.7 ± 0.3 µM), and eggplant (Ki: 4.8 ± 1.2 µM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 µM), mushroom (Ki: 567 ± 81 µM), and eggplant (Ki: 2016.7 ± 805.6 µM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.

Secretory Production of the Hericium erinaceus Laccase from Saccharomyces cerevisiae.

Mushroom laccases play a crucial role in lignin depolymerization, one of the most critical challenges in lignin utilization. Importantly, laccases can utilize a wide range of substrates, such as toxicants and antibiotics. This study isolated a novel laccase, named HeLac4c, from endophytic white-rot fungi Hericium erinaceus mushrooms. The cDNAs for this enzyme were 1569 bp in length and encoded a protein of 523 amino acids, including a 20 amino-acid signal peptide. Active extracellular production of glycosylated laccases from Saccharomyces cerevisiae was successfully achieved by selecting an optimal translational fusion partner. We observed that 5 and 10 mM Ca2+, Zn2+, and K+ increased laccase activity, whereas 5 mM Fe2+ and Al3+ inhibited laccase activity. The laccase activity was inhibited by the addition of low concentrations of sodium azide and L-cysteine. The optimal pH for the 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt was 4.4. Guaiacylglycerol-ß-guaiacyl ether, a lignin model compound, was polymerized by the HeLac4c enzyme. These results indicated that HeLac4c is a novel oxidase biocatalyst for the bioconversion of lignin into value-added products for environmental biotechnological applications.

Molecular basis of an atypical dsDNA 5mC/6mA bifunctional dioxygenase CcTet from Coprinopsis cinerea in catalyzing dsDNA 5mC demethylation.

The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes.

Azole inhibitors of mushroom and human tyrosinases: Current advances and prospects of drug development for melanogenic dermatological disorders.

Azoles are a famous and promising class of drugs for treatment of a range of ailments especially fungal infections. A wide variety of azole derivatives are also known to exhibit tyrosinase inhibition, some of which possess promising activity with potential for treatment of dermatological disorders such as post-inflammatory hyperpigmentation, nevus, flecks, melasma, and melanoma. Recently, thiazolyl-resorcinol derivatives have demonstrated potent human tyrosinase inhibition with a safe and effective therapeutic profile for treatment of skin hyperpigmentation in humans, which are currently under clinical trials. If approved these derivatives would be the first azole drugs to be used for treatment of skin hyperpigmentation. Although the scientific literature has been witnessing general reviews on tyrosinase inhibitors to date, there is none that specifically and comprehensively discusses azole inhibitors of tyrosinase. Appreciating such potential of azoles, this focused review highlights a wide range of their derivatives with promising mushroom and human tyrosinase inhibitory activities and clinical potential for treatment of melanogenic dermatological disorders. Presently, these disorders have been treated with kojic acid, hydroquinone and other drugs, the design and development of which are based on their ability to inhibit mushroom tyrosinase. The active sites of mushroom and human tyrosinases carry structural differences which affect substrate or inhibitor binding. For this reason, kojic acid and other drugs pose efficacy and safety issues since they were originally developed using mushroom tyrosinase and have been clinically used on human tyrosinase. Design and development of tyrosinase inhibitors should be based on human tyrosinase, however, there are challenges in obtaining the human enzyme and understanding its structure and function. The review discusses these challenges that encompass structural and functional differences between mushroom and human tyrosinases and the manner in which they are inhibited. The review also gauges promising azole derivatives with potential for development of drugs against skin hyperpigmentation by analyzing and comparing their tyrosinase inhibitory activities against mushroom and human tyrosinases, computational data, and clinical profile where available. It aims to lay groundwork for development of new azole drugs for treatment of skin hyperpigmentation, melanoma, and related dermatological disorders.

Flavonoids as tyrosinase inhibitors in in silico and in vitro models: basic framework of SAR using a statistical modelling approach.

Flavonoids are widely distributed in plants and constitute the most common polyphenolic phytoconstituents in the human diet. In this study, the in vitro inhibitory activity of 44 different flavonoids (1-44) against mushroom tyrosinase was studied, and an in silico study and type of inhibition for the most active compounds were evaluated too. Tyrosinase inhibitors block melanogenesis and take part in melanin production or distribution leading to pigmentation diseases. The in vitro study showed that quercetin was a competitive inhibitor (IC50=44.38 ± 0.13 µM) and achieved higher antityrosinase activity than the control inhibitor kojic acid. The in silico results highlight the importance of the flavonoid core with a hydroxyl at C7 as a strong contributor of interference with tyrosinase activity. According to the developed statistical model, the activity of molecules depends on hydroxylation at C3 and methylation at C8, C7, and C3 in the benzo-γ-pyrane ring of the flavonoids.

In silico analysis and characterization of medicinal mushroom cystathionine beta-synthase as an angiotensin converting enzyme (ACE) inhibitory protein.

Angiotensin-converting enzyme (ACE) regulates blood pressure and has been implicated in several conditions including lung injury, fibrosis and Alzheimer's disease. Medicinal mushroom Ganordema lucidum (Reishi) cystathionine beta-synthase (GlCBS) was previously reported to possess ACE inhibitory activities. However, the inhibitory mechanism of CBS protein remains unreported. Therefore, this study integrates in silico sequencing, structural and functional based-analysis, protein modelling, molecular docking and binding affinity calculation to elucidate the inhibitory mechanism of GlCBS and Lignosus rhinocerus (Tiger milk mushroom) CBS protein (LrCBS) towards ACE. In silico analysis indicates that CBSs from both mushrooms share high similarities in terms of physical properties, structural properties and domain distribution. Protein-protein docking analysis revealed that both GlCBS and LrCBS potentially modulate the C-terminal domain of ACE (C-ACE) activity via regulation of chloride activation and/or prevention of substrate entry. GICBS and LrCBS were also shown to interact with ACE at the same region that presumably inhibits the function of ACE.

Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase.

With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A-ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 µM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.

Urolithin and Reduced Urolithin Derivatives as Potent Inhibitors of Tyrosinase and Melanogenesis: Importance of the 4-Substituted Resorcinol Moiety.

We previously reported (E)-ß-phenyl-α,ß-unsaturated carbonyl scaffold ((E)-PUSC) played an important role in showing high tyrosinase inhibitory activity and that derivatives with a 4-substituted resorcinol moiety as the ß-phenyl group of the scaffold resulted in the greatest tyrosinase inhibitory activity. To examine whether the 4-substituted resorcinol moiety could impart tyrosinase inhibitory activity in the absence of the α,ß-unsaturated carbonyl moiety of the (E)-PUSC scaffold, 10 urolithin derivatives were synthesized. To obtain more candidate samples, the lactone ring in synthesized urolithins was reduced to produce nine reduced urolithins. Compounds 1c (IC50 = 18.09 ± 0.25 µM), 1h (IC50 = 4.14 ± 0.10 µM), and 2a (IC50 = 15.69 ± 0.40 µM) had greater mushroom tyrosinase-inhibitory activities than kojic acid (KA) (IC50 = 48.62 ± 3.38 µM). The SAR results suggest that the 4-substituted resorcinol motif makes an important contribution to tyrosinase inhibition. To investigate whether these compounds bind to human tyrosinase, a human tyrosinase homology model was developed. Docking simulations with mushroom and human tyrosinases showed that 1c, 1h, and 2a bind to the active site of both tyrosinases with higher binding affinities than KA. Pharmacophore analyses showed that two hydroxyl groups of the 4-substituted resorcinol entity act as hydrogen bond donors in both mushroom and human tyrosinases. Kinetic analyses indicated that these compounds were all competitive inhibitors. Compound 2a inhibited cellular tyrosinase activity and melanogenesis in α-MSH plus IBMX-stimulated B16F10 melanoma cells more strongly than KA. These results suggest that 2a is a promising candidate for the treatment of skin pigment disorders, and show the 4-substituted resorcinol entity importantly contributes to tyrosinase inhibition.

Ageritin from Pioppino Mushroom: The Prototype of Ribotoxin-Like Proteins, a Novel Family of Specific Ribonucleases in Edible Mushrooms.

Ageritin is a specific ribonuclease, extracted from the edible mushroom Cyclocybe aegerita (synonym Agrocybe aegerita), which cleaves a single phosphodiester bond located within the universally conserved alpha-sarcin loop (SRL) of 23-28S rRNAs. This cleavage leads to the inhibition of protein biosynthesis, followed by cellular death through apoptosis. The structural and enzymatic properties show that Ageritin is the prototype of a novel specific ribonucleases family named 'ribotoxin-like proteins', recently found in fruiting bodies of other edible basidiomycetes mushrooms (e.g., Ostreatin from Pleurotus ostreatus, Edulitins from Boletus edulis, and Gambositin from Calocybe gambosa). Although the putative role of this toxin, present in high amount in fruiting body (>2.5 mg per 100 g) of C. aegerita, is unknown, its antifungal and insecticidal actions strongly support a role in defense mechanisms. Thus, in this review, we focus on structural, biological, antipathogenic, and enzymatic characteristics of this ribotoxin-like protein. We also highlight its biological relevance and potential biotechnological applications in agriculture as a bio-pesticide and in biomedicine as a therapeutic and diagnostic agent.

Structure Analysis and Study of Biological Activities of Condensed Tannins from Bruguiera gymnorhiza (L.) Lam and Their Effect on Fresh-Cut Lotus Roots.

Bruguiera gymnorhiza (L.) Lam is a mangrove plant that spread in many parts of the world. Though mangrove plant polyphenols have been reported to exhibit many biological activities, little is known about mangrove plant tannins. To explore the application value of tannins from B. gymnorhiza, analyses on the structure and biological activity of condensed tannins (CTs) from Bruguiera gymnorhiza (L.) Lam were carried out. The results from 13C nuclear magnetic resonance (13C-NMR) and reversed-phase, high-performance liquid chromatography (RP-HPLC) showed that the CTs were dominated by procyanidins, with a small quantity of prodelphinidins and propelargonidins; and that the monomeric constituents of B. gymnorhiza tannins were catechin/epicatechin, gallocatechin/epigallocatechin and afzelechin/epiafzelechin. The CTs were reversible and mixed competitive inhibitors of tyrosinase and the 50% inhibiting concentration (IC50) was estimated to be 123.90 ± 0.140 µg/mL. The antioxidant activities of CTs from B. gymnorhiza leaves were evaluated, the IC50 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) (ABTS) scavenging activities were 88.81 ± 0.135 and 105.03 ± 0.130 µg/mL, respectively, and the ferric ion reducing antioxidant power (FRAP) value was 1052.27 ± 4.17 mgAAE/g. In addition, the results from fresh-keeping assays on fresh-cut lotus root reveal that CTs from B. gymnorhiza had excellent effects on inhibiting the activities of polyphenol oxidase (PPO) and peroxidase (POD), protecting fresh-cut lotus root from the oxidation of total phenolics and malondialdehyde (MDA) content and slowing the increase in total phenol content (TPC) at 4 °C during the whole storage period. Therefore, CTs showed good effects against the browning of fresh-cut lotus root. Together, these results suggested that B. gymnorhiza CTs are promising antibrowning agents for fresh-cut fruits.

Identification of Mushroom and Murine Tyrosinase Inhibitors from Achillea biebersteinii Afan. Extract.

Growing scientific evidence indicates that Achillea biebersteinii is a valuable source of active ingredients with potential cosmetic applications. However, the data on its composition and pharmacological properties are still insufficient. This study aims to optimize the extraction procedure of the plant material, evaluate its phytochemical composition, and compare anti-tyrosinase potential of A. biebersteinii extracts obtained by various methods. In order to identify compounds responsible for the tyrosinase inhibitory activity of A. biebersteinii, the most active anti-tyrosinase extract was fractionated by column chromatography. The fractions were examined for their skin lightening potential by mushroom and murine tyrosinase inhibitory assays and melanin release assay. HPLC-ESI-Q-TOF-MS/MS analysis of the total extract revealed the presence of several phenolic acids, flavonoids, flavonoid glucosides, and carboxylic acid. Among them, fraxetin-8-O-glucoside, quercetin-O-glucopyranose, schaftoside/isoschaftoside, gmelinin B, 1,3-dicaffeoylquinic acid (1,3-DCQA), and ferulic acid were found in the fractions with the highest skin lightening potential. Based on obtained qualitative and quantitative analysis of the fractions, it was assumed that the caffeoylquinic acid derivatives and dicaffeoylquinic acid derivatives are more likely responsible for mushroom tyrosinase inhibitory activity of A. biebersteinii extracts and fractions. Ferulic acid was proposed as the most active murine tyrosinase inhibitor, responsible also for the reduced melanin release from B16F10 murine melanoma cells.

Purification and characterization of a novel protein with activity against non-small-cell lung cancer in vitro and in vivo from the edible mushroom Boletus edulis.

A new anti-tumor protein (designated as Boletus edulis or in short BEAP) was isolated from dried fruit bodies of the edible bolete mushroom Boletus edulis. The purification protocol employed comprised fast ion exchange chromatography on a Hitrap Q column and ion exchange chromatography on a DEAE-52 cellulose column. Superdex G75 gel filtration and SDS-PAGE analysis revealed that BEAP was a protein with a molecular weight of 16.7 KD. The protein exhibited potent anti-cancer activity on A549 cells both in vitro and in vivo. With the use of AO/EB staining, annexin V-FITC/PI, and Western blotting, it was demonstrated in vitro that the cytotoxicity of BEAP was mediated by induction of apoptosis and arrest of A549 cells in the G1 phase of the cell cycle. BEAP significantly suppressed the growth of A549 solid tumors in vivo. These results prove that BEAP is a new multifunctional protein with anti-tumor and anti-metastasis capabilities.

Impacts of high pressure assisted freezing on the denaturation of polyphenol oxidase.

The mechanism of enzyme protein denaturation induced by high pressure freezing is complicated and unclear as this process involves Pressure-Factors (pressure and time) and Freezing-Factors (temperature, phase transition, recrystallization, and ice crystal types). In this study, the thermodynamics and conformation changes of mushroom polyphenol oxidase (PPO) under high pressure freezing treatments (HPF, 100,150,200,300,400,500MPaP-20°C/30min) and high pressure processes (HPP) followed with normal pressure immersion freezing (HPP-IF, 100-500MPaP25°C/30min - 0.1MPaP-20°C/30min) are investigated as compared with that processed under high pressure processes (HPP, 100-500MPaP25°C/30min) and normal pressure immersion freezing process (IF, 0.1MPaP-20°C/30min). The results suggested that the treated PPO with the same enzyme activity may have various thermodynamic characteristics and conformations; Pressure-Factors play the main roles in the denaturation of the PPO during the HPF treatment, and Freezing-Factors can weak the effect of Pressure-Factors on PPO denaturation; The treated PPO may be transferred into a partially fold intermediate state.

Inactivation of polyphenol oxidase by microwave and conventional heating: Investigation of thermal and non-thermal effects of focused microwaves.

Emerging technologies, such as focused microwave heating of liquid foods, have been studied to reduce quality losses due to the high temperatures of conventional processing. Besides faster heating, microwaves can also have non-thermal effects on inactivation; however, this is a controversial issue. The objective of this study was to compare conventional and focused microwave heating under similar conditions for the inactivation of two polyphenol oxidases (PPOs): mushroom tyrosinase in buffer and the PPO present in coconut water. Small samples under stirring were treated at temperatures between 50 and 90 °C and three kinetic models were adjusted considering the whole time-temperature history. The Weibull model could best describe inactivation in both heating processes, which was more effective with microwave heating for temperatures over 70 °C. Validation runs show that the model can satisfactorily describe the PPO inactivation. This study contributes for the design of liquid food pasteurization by focused microwave technology.

Effect of phenolic compounds from the leaves of Psidium guajava on the activity of three metabolism-related enzymes.

Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer); due to the numerous side effects of the latter. In vitro screening assays were conducted for Psidium guajava leaf methanolic extract against three metabolism-related enzymes; α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained weak and moderate multitarget inhibition against α-amylase, tyrosinase, and hyaluronidase, respectively; however, the leaf fractions exhibited stronger inhibitions for the three investigated enzymes. Fractionation of P. guajava leaf extract revealed that anthraquinones and ellagic acid are of the major active compounds with inhibitory activities for α-amylase, tyrosinase, and hyaluronidase. Kinetic studies showed that quinalizarin inhibition is competitive for both α-amylase and hyaluronidase, and ellagic acid inhibition for tyrosinase and hyaluronidase is competitive and un-competitive, respectively. The molecular docking studies of quinalizarin and ellagic acid with α-amylase, tyrosinase, and hyaluronidase showed high binding energies with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. guajava leaf fractions, quinalizarin and ellagic acid, have multitarget activities with potential therapeutic applications in many metabolic disorders.

Synthesis, antioxidant and anti-tyrosinase activity of 1,2,4-triazole hydrazones as antibrowning agents.

A series of 1,2,4-triazole hydrazones (1-16) were synthesized, and their inhibitory activities and mechanisms on tyrosinase were investigated by ultraviolet spectrophotometry, fluorescence quenching, molecular docking study, etc. Most of compounds possessed potent tyrosinase inhibitory activity. Thereinto, compound 9 presented the superior activity with IC50 of 0.9 µM, which was markedly lower than the standard kojic acid (IC50 = 64.1 µM). Compound 9 not only interacted with copper ions in the active center of the enzyme but also bound to the enzyme-substrate complex, indicating that it was a competitive-noncompetitive mixed inhibitor. Additionally, it also displayed potent DPPH scavenging activity. Antibrowning test showed that compound 9 effectively reduced the enzymatic browning of fresh-cut potatoes. Furthermore, compound 9 exhibited low cytotoxic activity against human normal cell line with IC50 of 49.9 µM. Overall, the present study suggests that these compounds may serve as lead molecules for developing novel antibrowning agents in food industry.

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates-namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases-we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways and mapped the appearance of the different enzyme types in a time-calibrated species tree.

Stabilization-destabilization and redox properties of laccases from medicinal mushroom Ganoderma lucidum and human pathogen Yersinia enterocolitica.

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature (<30 °C), while the isozymes were destabilized at neutral pH (7.0) and high-temperature conditions (>70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.

(E)-1-(Furan-2-yl)-(substituted phenyl)prop-2-en-1-one Derivatives as Tyrosinase Inhibitors and Melanogenesis Inhibition: An In Vitro and In Silico Study.

A series of (E)-1-(furan-2-yl)prop-2-en-1-one derivatives (compounds 1-8) were synthesized and evaluated for their mushroom tyrosinase inhibitory activity. Among these series, compound 8 (2,4-dihydroxy group bearing benzylidene) showed potent tyrosinase inhibitory activity, with respective IC50 values of 0.0433 µM and 0.28 µM for the monophenolase and diphenolase as substrates in comparison to kojic acid as standard compound 19.97 µM and 33.47 µM. Moreover, the enzyme kinetics of compound 8 were determined to be of the mixed inhibition type and inhibition constant (Ki) values of 0.012 µM and 0.165 µM using the Lineweaver-Burk plot. Molecular docking results indicated that compound 8 can bind to the catalytic and allosteric sites 1 and 2 of tyrosinase to inhibit enzyme activity. The computational molecular dynamics analysis further revealed that compound 8 interacted with two residues in the tyrosinase active site pocket, such as ASN260 and MET280. In addition, compound 8 attenuated melanin synthesis and cellular tyrosinase activity, simulated by α-melanocyte-stimulating hormone and 1-methyl-3-isobutylxanthine. Compound 8 also decreased tyrosinase expressions in B16F10 cells. Based on in vitro and computational studies, we propose that compound 8 might be a worthy candidate for the development of an antipigmentation agent.