Functional characterization of polyphenol oxidase OfPPO2 supports its involvement in parallel biosynthetic pathways of acteoside.

Acteoside is a bioactive phenylethanoid glycoside widely distributed throughout the plant kingdom. Because of its two catechol moieties, acteoside displays a variety of beneficial activities. The biosynthetic pathway of acteoside has been largely elucidated, but the assembly logic of two catechol moieties in acteoside remains unclear. Here, we identified a novel polyphenol oxidase OfPPO2 from Osmanthus fragrans, which could hydroxylate various monophenolic substrates, including tyrosine, tyrosol, tyramine, 4-hydroxyphenylacetaldehyde, salidroside, and osmanthuside A, leading to the formation of corresponding catechol-containing intermediates for acteoside biosynthesis. OfPPO2 could also convert osmanthuside B into acteoside, creating catechol moieties directly via post-modification of the acteoside skeleton. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and subcellular localization assay further support the involvement of OfPPO2 in acteoside biosynthesis in planta. These findings suggest that the biosynthesis of acteoside in O. fragrans may follow "parallel routes" rather than the conventionally considered linear route. In support of this hypothesis, the glycosyltransferase OfUGT and the acyltransferase OfAT could direct the flux of diphenolic intermediates generated by OfPPO2 into acteoside. Significantly, OfPPO2 and its orthologs constitute a functionally conserved enzyme family that evolved independently from other known biosynthetic enzymes of acteoside, implying that the substrate promiscuity of this PPO family may offer acteoside-producing plants alternative ways to synthesize acteoside. Overall, this work expands our understanding of parallel pathways plants may employ to efficiently synthesize acteoside, a strategy that may contribute to plants' adaptation to environmental challenges.

The PPO family in Nicotiana tabacum is an important regulator to participate in pollination.

Polyphenol oxidases (PPOs) are type-3 copper enzymes and are involved in many biological processes. However, the potential functions of PPOs in pollination are not fully understood. In this work, we have screened 13 PPO members in Nicotiana. tabacum (named NtPPO1-13, NtPPOs) to explore their characteristics and functions in pollination. The results show that NtPPOs are closely related to PPOs in Solanaceae and share conserved domains except NtPPO4. Generally, NtPPOs are diversely expressed in different tissues and are distributed in pistil and male gametes. Specifically, NtPPO9 and NtPPO10 are highly expressed in the pistil and mature anther. In addition, the expression levels and enzyme activities of NtPPOs are increased after N. tabacum self-pollination. Knockdown of NtPPOs would affect pollen growth after pollination, and the purines and flavonoid compounds are accumulated in self-pollinated pistil. Altogether, our findings demonstrate that NtPPOs potentially play a role in the pollen tube growth after pollination through purines and flavonoid compounds, and will provide new insights into the role of PPOs in plant reproduction.

Mushroom Tyrosinase: Six Isoenzymes Catalyzing Distinct Reactions.

"Mushroom tyrosinase" from the common button mushroom is the most frequently used source of tyrosinase activity, both for basic and applied research. Here, the complete tyrosinase family from Agaricus bisporus var. bisporus (abPPO1-6) was cloned from mRNA and expressed heterologously using a single protocol. All six isoenzymes accept a wide range of phenolic and catecholic substrates, but display pronounced differences in their specificity and enzymatic reaction rate. AbPPO3 ignores γ-l-glutaminyl-4-hydroxybenzene (GHB), a natural phenol present in mM concentrations in A. bisporus, while AbPPO4 processes 100 µM GHB at 4-times the rate of the catechol l-DOPA. All six AbPPOs are biochemically distinct enzymes fit for different roles in the fungal life cycle, which challenges the traditional concept of isoenzymes as catalyzing the same physiological reaction and varying only in secondary properties. Transferring this approach to other enzymes and organisms will greatly stimulate both the study of the in vivo function(s) of enzymes and the application of these highly efficient catalysts.

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.

Feeding alfalfa-grass or red clover-grass mixture baleage: Effect on milk yield and composition, ruminal fermentation and microbiota taxa relative abundance, and nutrient utilization in dairy cows.

Our goal was to investigate the effect of diets containing baleages harvested from alfalfa-grass or red clover-grass mixture on production performance, ruminal fermentation and microbiota taxa relative abundance, milk fatty acid profile, and nutrient utilization in dairy cows. Twenty Jersey cows (18 multiparous and 2 primiparous) averaging (mean ± SD) 148 ± 45.2 days in milk and 483 ± 65.4 kg of body weight in the beginning of the study were used in a randomized complete block design with repeated measures over time. The experiment lasted 9 wk, with a 2 wk covariate period followed by 7 wk of data and sample collection (wk 4 and 7 used in the statistical analyses). Cows were fed diets containing (dry matter basis) 35% of a concentrate mash and the following forage sources: (1) 65% second- and third-cut (32.5% each) alfalfa-grass mixture baleages (ALF) or (2) 65% second- and third-cut (32.5% each) red clover-grass mixture baleages (RC). Diets did not affect dry matter intake, milk yield, and concentrations of milk fat and true protein. In contrast, milk fat yield tended to decrease and energy-corrected milk yield decreased with feeding RC versus ALF. The apparent total-tract digestibilities of dry matter, organic matter, and ash-free neutral detergent fiber, milk proportions of trans-10 18:1, cis-9,cis-12,cis-15 18:3, and total n-3 fatty acids, ruminal molar proportion of acetate, and plasma concentrations of Leu, Phe, and Val all increased in RC versus ALF. Diet × week interactions were found for several parameters, most notably ruminal molar proportions of propionate and butyrate, ruminal NH3-N, milk urea N, plasma urea N, and plasma His concentrations, urinary N excretion, enteric CH4 production, and all energy efficiency variables. Specifically, ruminal NH3-N and plasma urea N concentrations, urinary excretion of N, and CH4 production decreased in cows fed RC in wk 4 but not in wk 7. Milk urea N concentration decreased and that of plasma His increased with feeding RC during wk 4 and 7, although the magnitude of treatments difference varied between the sampling periods. Efficiency of energy utilization calculated as milk energy/metabolizable energy decreased and that of tissue energy/ME increased in RC versus ALF cows in wk 4, suggesting that ME was portioned toward tissue and not milk in the RC diet. Interactions were also observed for the relative abundance of the rumen bacterial phyla Verrucomicrobiota and Fibrobacterota, with cows offered RC showing greater values than those receiving ALF in wk 4 but no differences in wk 7. Several diet × week interactions were detected in the present study implying short-term treatment responses and warranting further investigations.

Specific Substrate Activity of Lotus Root Polyphenol Oxidase: Insights from Gaussian-Accelerated Molecular Dynamics and Markov State Models.

Polyphenol oxidase (PPO) plays a key role in the enzymatic browning process, and this study employed Gaussian-accelerated molecular dynamics (GaMD) simulations to investigate the catalytic efficiency mechanisms of lotus root PPO with different substrates, including catechin, epicatechin, and chlorogenic acid, as well as the inhibitor oxalic acid. Key findings reveal significant conformational changes in PPO that correlate with its enzymatic activity. Upon substrate binding, the alpha-helix in the Q53-D63 region near the copper ion extends, likely stabilizing the active site and enhancing catalysis. In contrast, this helix is disrupted in the presence of the inhibitor, resulting in a decrease in enzymatic efficiency. Additionally, the F350-V378 region, which covers the substrate-binding site, forms an alpha-helix upon substrate binding, further stabilizing the substrate and promoting catalytic function. However, this alpha-helix does not form when the inhibitor is bound, destabilizing the binding site and contributing to inhibition. These findings offer new insights into the substrate-specific and inhibitor-induced structural dynamics of lotus root PPO, providing valuable information for enhancing food processing and preservation techniques.

Purification and Biochemical Characterization of Polyphenol Oxidase Extracted from Wheat Bran (Wan grano).

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL-1·min-1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of -5.28 kcal/mol, -4.65 kcal/mol, -4.21 kcal/mol, and -5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea.

Polyphenol Oxidase Activity on Guaiacyl and Syringyl Lignin Units.

The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant-based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose-degrading ascomycetes for their capacity to react with G-type and S-type phenolic compounds. All six PPOs catalyze the ortho-hydroxylation of G-type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy-ortho-diphenols. Remarkably, a subset of these PPOs is also active towards S-compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy-ortho-diphenols. Assays with 18O2 demonstrate that these PPOs in particular catalyze ortho-hydroxylation and ortho-demethoxylation of S-compounds and generate methanol as a co-product. Notably, oxidative (ortho-)demethoxylation of S-compounds is a novel reaction for PPOs, which we propose occurs via a distinct reaction mechanism compared to aryl-O-demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy-ortho-diphenols from both G- and S-type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin-derived compounds.

Purification, characterization and inactivation kinetics of polyphenol oxidase extracted from Cistanche deserticola.

MAIN CONCLUSION: PPO was purified from Cistanche deserticola, and its enzymatic characteristics were clarified. It was found that microwave treatment was an efficient way to inactivate PPO. Polyphenol oxidase (PPO) from Cistanche deserticola was obtained and purified through an acetone precipitation and anion exchange column, the enzymatic characteristics and inactivation kinetics of PPO were studied. The specific activity of PPO was 73135.15 ± 6625.7 U/mg after purification, the purification multiple was 48.91 ± 4.43 times, and the recovery was 30.96 ± 0.27%. The molecular weight of the PPO component is about 66 kDa by SDS-PAGE analysis. The optimum substrate of PPO was catechol (Vmax = 0.048 U/mL, Km = 21.70 mM) and the optimum temperature and pH were 30 °C and 7, respectively. When the temperature is above 50 °C, pH < 3 or pH > 10, the enzyme activity can be significantly inhibited. The first-order kinetic fitting shows that microwave inactivation has lesser k values, larger D values and shorter t1/2. It was found that microwave treatment is considered as an efficient and feasible way to inactive PPO by comparing the Z values and Ea values of the two thermal treatments.

Enhancing defense against rice blast disease: Unveiling the role of leaf endophytic firmicutes in antifungal antibiosis and induced systemic resistance.

Rice remains the primary staple for more than half of the world's population, yet its cultivation faces numerous challenges, including both biotic and abiotic stresses. One significant obstacle is the prevalence of rice blast disease, which substantially diminishes productivity and increases cultivation costs due to frequent fungicide applications. Consequently, the presence of fungicide residues in rice raises concerns about compliance with international maximum residue limits (MRLs). While host resistance has proven effective, it often remains vulnerable to new variants of the Magnaporthe oryzae pathogen. Therefore, there is a critical need to explore innovative management strategies that can complement or enhance existing methods. An unexplored avenue involves harnessing endophytic bacterial communities. To this end, the present study investigates the potential of eleven endophytic Bacillus spp. in suppressing Pyricularia oryzae, promoting plant growth, and eliciting a defense response through phyllobacterization. The results indicate that the secreted metabolome and volatilome of seven tested isolates demonstrate inhibitory effects against P.oryzae, ranging from a minimum of 40% to a maximum of 70%. Bacillus siamensis L34, B. amyloliquefaciens RA37, B. velezensis L12, and B. subtilis B18 produce antifungal antibiotics targeting P.oryzae. Additionally, B. subtilis S4 and B. subtilis S6 emerge as excellent inducers of systemic resistance against blast disease, as evidenced by elevated activity of biochemical defense enzymes such as peroxidase, polyphenol oxidase, and total phenol content. However, a balance between primary metabolic activity (e.g., chlorophyll content, chlorophyll fluorescence, and photosynthetic rate) and defense activity is observed. Furthermore, specific endophytic Bacillus spp. significantly stimulates defense-related genes, including OsPAD4, OsFMO1, and OsEDS1. These findings underscore the multifaceted potential of endophytic Bacillus in managing blast disease through antibiosis and induced systemic resistance. In conclusion, this study highlights the promising role of endophytic Bacillus spp. as a viable option for blast disease management. Their ability to inhibit the pathogen and induce systemic resistance makes them a valuable addition to the existing strategies. However, it is crucial to consider the trade-off between primary metabolic activity and defense response when implementing these bacteria-based approaches.