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The rise in industrial establishments all around the globe has also enhanced the demand of enzymes for usage in processes. Laccases are one of the dominant enzymes which have been employed in industrial processes over the years. They are a type of multi-copper oxidases (MCOs) which is a category of enzymes having diverse substrate selectivity and varying biological activity. All multi-copper oxidases can convert oxygen into water without creating unwanted products owing to the existence of cupredoxin-like domains. This review article details up-to-date knowledge about laccases, their structures and various sources, the laccase mediator system, mechanism of action and applications of laccases in numerous biotechnological processes such as in food industry for wine and beer stabilization, synthesis of cosmetics and pharmaceutically important compounds, dye detoxification in industrial effluents, xenobiotic degradation, paper and pulp bleaching, biofuel cells and biosensor development for detection for various chemical compounds. [ABSTRACT FROM AUTHOR]

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min−1 mM−1. Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds. Key points: • Identification and characterization of two novel bacterial aryl-alcohol oxidases • Crystal structures reveal wide-open active-site pockets, impacting substrate scope • Total turnover numbers and cell-free extracts demonstrate the synthetic potential [ABSTRACT FROM AUTHOR]

Summary Reactive oxygen species (ROS) are among the most important signaling molecules, playing a significant role in plant growth, development, and responses to various environmental stresses. Respiratory burst oxidase homologs (RBOHs) are key enzymes in ROS production. Plants tightly regulate the activation and deactivation of RBOHs through various post‐translational modifications (PTMs), including phosphorylation, ubiquitination, S‐nitrosylation, and persulfidation. These PTMs fine‐tune ROS production, ensuring normal plant growth and development while facilitating rapid responses to abiotic and biotic stresses. This review discusses the effects of different PTMs on RBOH function and their biological relevance. Additionally, we examine the evolutionary conservation of PTM sites and emphasize the complex interplay between multiple PTMs regulating RBOHs. [ABSTRACT FROM AUTHOR]

The integration of organocatalysis and enzyme catalysis in one‐pot cascade processes allows for the efficient construction of complex molecular architectures with high levels of stereocontrol. However, challenges related to reaction compatibility between both processes are often a limitation for the development of efficient synthetic routes. In this study, we describe the combination of an enzymatic aerobic oxidation followed by the squaramide‐mediated asymmetric formation of C−P and C−C bonds to access important building blocks such as chiral α‐hydroxyphosphonates and β‐nitro alcohols in good yields and enantiomeric ratios. This sequential process is conducted in a one‐pot fashion within a biphasic system and represents a pioneering example of a chemoenzymatic cascade involving aerobic biooxidation and an organocatalytic step operating under hydrogen‐bond activation mode and under mild reaction conditions. [ABSTRACT FROM AUTHOR]

The aging process is influenced by the accumulation of both beneficial and harmful factors in the external environment. While extensive literature has explored the health benefits of short-term forest bathing, the potential for long-term exposure to forests to delay the aging process remains unclear. This study aimed to investigate the impact of prolonged urban forest contact on aging using a murine model. Forty male Specified Pathogen Free degree Kunming mice (6 weeks old, average weight 30.44 ± 0.91 g) were subjected to continuous subcutaneous injection of D-galactose in an urban environment and an urban forest environment to simulate an accelerated aging process lasting six weeks. Results showed that compared to urban environment, mice living in the forest environment exhibited significantly increased levels of serum oxidases CAT, GSH-Px, and SOD, as well as decreased levels of oxidative products H2O2 and MDA. These findings suggest that long-term forest living enhances serum Nrf2 expression, downstream oxidase activity, total antioxidant capacity, and resistance to free radicals and oxidative products, thus delaying the aging process. This study offers valuable insights into the potential health benefits of long-term forest living, which may play a role in delaying the aging process in humans. [ABSTRACT FROM AUTHOR]

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri , M. browni , and M. melanurus , of which the venom composition of M. melanurus , the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni , and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri , and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A 2 (PLA 2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l -amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri , on the other hand, remains necessary. [Display omitted] • The first proteomic characterization of Mixcoatlus browni and M. barbouri venoms is reported. • A new Crotoxin-like protein in the venom of Mixcoatlus browni is characterized. • Mixcoatlus barbouri venom contains homologs of acidic and basic subunits of Crotoxin. • The Antivipmyn antivenom can neutralize the lethality of M. browni. [ABSTRACT FROM AUTHOR]

A study on influence of nutrient factors and defensive enzymes on maize hybrids was conducted where, nitrogen, phosphorous, potassium and peroxidases, polyphenol oxidases were analysed, respectively. Nitrogen and phosphorous increased susceptibility of maize hybrids to corn leaf aphid infestation whereas, potassium, peroxidase and polyphenol oxidase did not favour the infestation. Nutritional components in leaf samples of maize hybrids on the infestation of corn leaf aphid revealed that the infestation was significant and correlated positively with nitrogen (0.953**) and phosphorus (0.986**). While a significant and negative correlation was observed in case of potassium (-0.952**), peroxidase (-0.980*) and polyphenol oxidase (-0.822**). Whereas, nutritional components in tassels of maize hybrids on the infestation of corn leaf aphid was significant and correlated positively with nitrogen (0.976**) and phosphorus (0.938**). While a significant negative correlation was observed in case of potassium (-0.838**), peroxidase (-0.873*) and polyphenol oxidase (-0.968**). [ABSTRACT FROM AUTHOR]

In plants, a local infection can lead to systemic acquired resistance (SAR) through increased production of salicylic acid (SA). For many years, the identity of the mobile signal and its direct transduction mechanism for systemic SA synthesis in initiating SAR have been debated. We found that in Arabidopsis thaliana, after a local infection, the conserved cysteine residue of the transcription factor CCA1 HIKING EXPEDITION (CHE) undergoes sulfenylation in systemic tissues, which enhances its binding to the promoter of the SA-synthesis gene ISOCHORISMATE SYNTHASE1 (ICS1) and increases SA production. Furthermore, hydrogen peroxide (H2O2) produced through NADPH oxidases is the mobile signal that sulfenylates CHE in a concentration-dependent manner. Accumulation of SA and the previously reported signal molecules, such as N-hydroxypipecolic acid (NHP), then form a signal amplification loop to establish SAR. [ABSTRACT FROM AUTHOR]

The preharvest internal browning of Nane plum fruit, with no visible effects on the appearance of the fruit, has become a serious problem in recent years in its production area in Guangdong Province, China. This study investigated the effects of environmental factors, including temperature, on Nane plum internal browning. Plum orchards at different elevations with different incidences of internal browning were selected. Using fruits with different internal browning incidence levels, the internal browning mechanism was analyzed with transcriptome and metabolome analyses. The results revealed decreased internal browning at high altitudes. Shading treatment significantly reduced internal browning, whereas bagging and insect-proof net-covering treatments significantly increased internal browning. Because bagging and net coverings increase the local ambient temperature, the findings suggest that high temperature is an important factor influencing the internal browning of Nane plum. The metabolome experiments showed that with increased internal browning, the levels of phenolic hydroxyls such as catechol increased, with simultaneous increases in hydrogen peroxide content and oxidase activity. It can be speculated that the oxidation of phenolic hydroxyl substances is the main cause of the preharvest browning of Nane plum. Transcriptome analysis revealed the increased expression of calcium signaling-related and downstream effector genes and indicated an important role of calcium in internal browning, possibly due to its increased content in the fruit. Further, with increasingly serious internal browning, genes related to photosynthesis were down-regulated, while genes related to senescence were up-regulated, thus suggesting the up-regulation of the process of cell senescence during internal browning. In conclusion, heat stress should be eliminated to reduce preharvest internal browning in Nane plum. [ABSTRACT FROM AUTHOR]

Japanese green tea, an essential beverage in Japanese culture, is characterized by the initial steaming of freshly harvested leaves during production. This process efficiently inactivates endogenous enzymes such as polyphenol oxidases, resulting in the production of sencha, gyokuro and matcha that preserves the vibrant green color of young leaves. Although genome sequences of several tea cultivars and germplasms have been published, no reference genome sequences are available for Japanese green tea cultivars. Here, we constructed a reference genome sequence of the cultivar 'Seimei', which is used to produce high-quality Japanese green tea. Using the PacBio HiFi and Hi-C technologies for chromosome-scale genome assembly, we obtained 15 chromosome sequences with a total genome size of 3.1 Gb and an N50 of 214.9 Mb. By analyzing the genomic diversity of 23 Japanese tea cultivars and lines, including the leading green tea cultivars 'Yabukita' and 'Saemidori', it was revealed that several candidate genes could be related to the characteristics of Japanese green tea. The reference genome of 'Seimei' and information on genomic diversity of Japanese green tea cultivars should provide crucial information for effective breeding of such cultivars in the future. [ABSTRACT FROM AUTHOR]

Two copper (II) complexes based on the mixed ligand system were synthesized and structurally characterized. The molecular formulae for complex (1), [Cu2L1L′] and complex (2), [CuL2L′] were determined based on analytical data, molar conductance, and thermal analysis results. The optimized structures of complexes (1) and (2) were determined by magnetic and spectroscopic measurements (FTIR, UV–Vis, and ESR) and confirmed by processing powder X‐ray diffraction data by Expo 2014 software. The geometry index (τ5), determined by ESR spectra and structural analysis based on powder X‐ray, demonstrates the square pyramidal geometry of the Cu (II) center of [Cu2L1L′] and [CuL2L′]. Cyclic voltammetry measurements were performed to elucidate the relationship between the redox properties of (1) and (2) and their oxidase‐mimicking catalytic activity. Catalytic aerobic oxidation of the polyphenols 3,5‐di‐tert‐butylcatechol (3,5‐DTBCH2), 4‐nitrocatechol, and 2‐aminophenol (2‐APH3) was studied by stopped‐flow technique. Promising catechol oxidase (COX) and phenoxazinone synthase (PHS) mimetic activity have been demonstrated for (1) and (2). Electrochemical data were used to determine the free energy (ΔG°) or driving force (−λ), and their correlation with the oxidase mimetic activity of(1) and (2) was examined. Several experimental procedures have been performed that have helped describe the catalytic mechanisms of the catalytic reactions taking place. [ABSTRACT FROM AUTHOR]

Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one‐pot synthesis of 2‐aryl thiazolines from various 4‐hydroxybenzaldehydes and aminothiols. The wild‐type biocatalyst features a broad scope of 4‐hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi‐rational protein engineering, generating a variant to produce previously inaccessible cysteine‐derived bioactive 2‐aryl thiazolines using the wild‐type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2‐aryl‐thiazoline derivatives to prompt their use in drug discovery and catalysis. [ABSTRACT FROM AUTHOR]

The study aimed to assess the nutritional properties of fruit juices using the microwave flow pasteurization (MFP) method. The innovative spray deaeration process was also employed at two different temperatures, 25 °C and 50 °C, with three rotational speeds for the spray nozzle: 150, 450, and 750 rpm. The research focused on two not-from-concentrate (NFC) juices: apple–peach and apple–chokeberry. The innovative MFP method demonstrated significant results over 12 months of storage: no presence of Listeria monocytogenes or Salmonella spp. bacteria was detected. Polyphenol oxidases (PPOs) were inactivated, while peroxidase (POD) activity in apple–peach juice was minimal. The total polyphenol content (TPC) in the juices gradually decreased over storage time, but MFP resulted in a slower degradation of TPC than traditional pasteurization (TP). Additionally, anthocyanin and carotenoid content gradually decreased during storage time. Still, with MFP, higher concentrations of these compounds were noted up to 4 and 6 months of storage in apple–peach and apple–chokeberry juices, respectively, than with TP. The research findings indicate that MFP may be a suitable and promising technique for preserving high-quality juices with superior retention of essential nutrients. However, the recommended storage time should be at most four months. [ABSTRACT FROM AUTHOR]

In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes. Single-atom nanozymes are a type of non-protein artificial enzymes and promising for mimicking enzyme active centers, but lack recognition sites to confer substrate specificity. Here, the authors report on a metal-ligand dual-site single-atom nanozyme (Ni-DAB) that mimics the dual-site catalytic mechanism of urate oxidase and has high selectivity in uric acid (UA) oxidation. [ABSTRACT FROM AUTHOR]

Pleurotus ostreatus is one of the most consumed mushroom species, as it serves as a high-quality food, favors a rich secondary metabolism, and has remarkable adaptability to the environment and predators. In this study, we investigated the function of two key reactive oxygen species producing enzyme NADPH oxidase (PoNoxA and PoNoxB) in P. ostreatus hyphae growth, metabolite production, signaling pathway activation, and immune responses to different stresses. Characterization of the Nox mutants showed that PoNoxB played an important role in the hyphal formation of the multicellular structure, while PoNoxA regulated apical dominance. The ability of P. ostreatus to tolerate a series of abiotic stress conditions (e.g., osmotic, oxidative, membrane, and cell-wall stresses) and mechanical damage repair was enhanced with PoNoxA over-expression. PoNoxB had a greater responsibility in regulating the polysaccharide composition of the cell wall and methyl jasmonate and gibberellin GA1 biosynthesis, and improved mushroom resistance against Tyrophagus putrescentiae. Moreover, mutants were involved in the jasmonate and GA signaling pathway, and toxic protein defense metabolite production. Our findings shed light on how the oyster mushroom senses stress signals and responds to adverse environments by the complex regulators of Noxs. [ABSTRACT FROM AUTHOR]

Nanozymes are widely used in various applications as nanosized catalysts for replacing enzymes. An accurate estimation of the catalytic activity of nanozymes in real conditions is critical. In this article, for the first time, we systematically studied the effect of macromolecular molecules co-existing in the real system on the oxidoreductase (peroxidase, oxidase, and catalase)-mimicking nanozymes made of a gold nanoparticle core and a platinum shell, Prussian Blue, Mn2O3 and CoO nanoparticles. Comparisons were made with horseradish peroxidase. We distinguished two main mechanisms of the negative impact of macromolecules on nanozyme catalysis–slowed diffusion and surface shielding of nanoparticles. While the first mechanism is typical for enzymes, the second one is specific only for nanozymes. Understanding the mechanisms is essential for developing approaches to reduce the unavoidable impact of macromolecules for various analytical and biomedical applications. [ABSTRACT FROM AUTHOR]

Coriolopsis trogii is a basidiomycete fungus which utilizes a large array of lignin-modifying enzymes to colonize and decompose dead wood. Its extracellular enzymatic arsenal includes laccases, i.e., polyphenol oxidases of relevant interest for different industrial applications thanks to their ability to oxidize a diverse range of natural and synthetic compounds. In this work, the production of laccases in C. trogii MUT3379 was explored and improved. From an initial production of ca. 10,000 U L−1, the fermentation process was gradually optimized, reaching a final yield of ca. 200,000 U L−1. An SDS-PAGE analysis of the secretome highlighted the presence of a main protein of ca. 60 kDa showing laccase activity, which was designated as Lac3379-1 once its primary sequence was established by tandem mass spectrometry. The characterization of Lac3379-1 revealed a remarkable enzymatic stability in the presence of surfactants and solvents and a diversified activity on a broad range of substrates, positioning it as an interesting tool for diverse biotechnological applications. The high-yield and robust production process indicates C. trogii MUT3379 as a promising cell factory for laccases, offering new perspectives for industrial applications of lignin-modifying enzymes. [ABSTRACT FROM AUTHOR]

Oxidation reactions catalysed by O2‐dependent enzymes are gaining increasing interest in the chemical industry due to their potential to provide a more selective and sustainable alternative to conventional chemical oxidation methods. O2‐dependent enzymes, like oxidases and oxygenases, catalyse a versatile range of oxidative reactions using only molecular oxygen as an oxidant. However, their practical application on a larger scale has been limited up to this point, primarily due to factors like their low catalytic rates combined with a narrow substrate spectrum and low stability. Nonetheless, enzyme engineering studies have significantly addressed these challenges in recent years and moved O2‐dependent enzymes closer towards industrial utilisation. This review aims to concisely overview the most recent engineering approaches to O2‐dependent enzymes. We will highlight recent studies that have targeted various aspects of O2‐dependent enzymes, including activity, selectivity, stability, and substrate spectrum, focusing on engineering studies where the engineered enzymes catalysed synthetically valuable reactions. [ABSTRACT FROM AUTHOR]

Background: Triple-negative breast cancer (TNBC) cells are a highly formidable cancer to treat. Nonetheless, by continued investigation into the molecular biology underlying the complex regulation of TNBC cell activity, vulnerabilities can be exposed as potential therapeutic targets at the molecular level. We previously revealed that lysyl oxidase-like 4 (LOXL4) promotes the invasiveness of TNBC cells via cell surface annexin A2 as a novel binding substrate of LOXL4, which promotes the abundant localization of integrin-b1 at the cancer plasma membrane. However, it has yet to be uncovered how the LOXL4-mediated abundance of integrin-b1 hastens the invasive outgrowth of TNBC cells at the molecular level. Methods: LOXL4-overexpressing stable clones were established from MDA-MB231 cells and subjected to molecular analyses, real-time qPCR and zymography to clarify their invasiveness, signal transduction, and matrix metalloprotease (MMP) activity, respectively. Results: Our results show that LOXL4 potently promotes the induction of matrix metalloprotease 9 (MMP9) via activation of nuclear factor-kB (NF-kB). Our molecular analysis revealed that TNF receptor-associated factor 4 (TRAF4) and TGF-b activated kinase 1 (TAK1) were required for the activation of NF-kB through Ikb kinase kinase (IKKa/b) phosphorylation. Conclusion: Our results demonstrate that the newly identified LOXL4-mediated axis, integrin-b1-TRAF4-TAK1-IKKa/b-Ikba-NF-kB-MMP9, is crucial for TNBC cell invasiveness. [ABSTRACT FROM AUTHOR]

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8‐dihydroxynaphthalene to generate the spiroketal linkage and 2,3‐epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C‐5 hydroxy group, widely found in SBNs. PalC catalyzes 1‐keto reduction, the reverse 1‐dehydrogenation, and 2,3‐epoxide reduction. Moreover, an FAD‐dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1‐dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs. [ABSTRACT FROM AUTHOR]

Enzymes are natures' catalysts and will have a lasting impact on (organic) synthesis as they possess unchallenged regio- and stereo selectivity. On the downside, this high selectivity limits enzymes' substrate range and hampers their universal application. Therefore, substrate scope expansion of enzyme families by either modification of known biocatalysts or identification of new members is a key challenge in enzyme-driven catalysis. Here, we present a streamlined approach to rationally select enzymes with proposed functionalities from the ever-increasing amount of available sequence data. In a case study on 4-phenol oxidoreductases, eight enzymes of the oxidase branch were selected from 292 sequences on basis of the properties of first shell residues of the catalytic pocket, guided by the computational tool A2CA. Correlations between these residues and enzyme activity yielded robust sequence-function relations, which were exploited by site-saturation mutagenesis. Application of a peroxidase-independent oxidase screening resulted in 16 active enzyme variants which were up to 90-times more active than respective wildtype enzymes and up to 6-times more active than the best performing natural variants. The results were supported by kinetic experiments and structural models. The newly introduced amino acids confirmed the correlation studies which overall highlights the successful logic of the presented approach. Enzymes play an important role in organic synthesis thanks to their high selectivity, however, such selectivity limits their substrate scope and hampers their wide application. Here, the authors report an approach to rationally select enzymes with proposed functionalities based on sequence analysis, exploring the substrate scope of 4-phenol oxidases by in silico sequence-function correlation analysis. [ABSTRACT FROM AUTHOR]

This study investigates the differential browning mechanisms among various cultivars of apples. The biochemical mechanism responsible for browning activities in apples involves a group of enzymes known as polyphenol oxidases (PPO). These enzymes facilitate the reaction between polyphenolic substrates (PPS) and oxygen in the production of benzoquinones, a compound that auto-polymerizes to form melanin, the primary browning agent in apples. In this study, both the PPO and PPS were extracted from five distinct apple cultivars, namely Fuji, Red Delicious, Gold Rush, Ruby Frost, and Mutsu. To identify the types of PPO in each subspecies, the reactivity of the extracted enzyme from each cultivar was quantified in the presence of three known substrates, catechol, catechin, and chlorogenic acid using UV-vis absorption spectrophotometry. The reactivity of the PPO extract with each substrate was compared across the five apple cultivars to identify the specificity of PPO to type(s) of PPS present and to quantify the concentration of PPO present in each species. Browning activity directly affects the longevity of fruits and vegetables, which is a consistent problem in the agricultural industry. If the biochemical mechanism of browning activity is better understood, it could lead to improvements in future food production, storage, and transportation methods. [ABSTRACT FROM AUTHOR]

The functional difference among the three copper-transporting P-type ATPases (CtpA, CtpB, and CtpV) annotated in genome of Mycobacterium tuberculosis (Mtb) remains unclear. Thus, CtpA and CtpB are believed to deliver copper to extracytoplasmic proteins as a cofactor required to overcome redox and copper stress in the Mtb periplasm. This study investigates an alternative role of CtpA-mediated copper transportation and its possible interaction with the activity of the multicopper oxidase, (MmcO), in response to redox stress. Results from RT-qPCR experiments indicate that the ctpA gene is upregulated in low Cu2+ concentrations, and under oxidative (H 2 O 2) and nitrosative (sodium nitroprusside) conditions in vitro, but not in high doses of Cu2+. Furthermore, the ctpA mutant strain (Mtb Δ ctpA) showed impaired growth in the presence of oxidative and nitrosative stress in vitro. However, it did not display such growth impairments in response to high doses of copper in comparison to the wild-type strain. Disruption of the ctpA gene in the Mtb genome did not induce an accumulation of copper in cells under toxic doses of the metal, suggesting that CtpA is not directly involved in copper detoxification. On the other hand, whole-cell lysates of the Mtb Δ ctpA mutant that were previously stimulated with Cu2+, H 2 O 2 and SNP (sodium nitroprusside), displayed reduced ability to oxidize organic substrates (para-phenylenediamine (pPD) and 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid) (ABTS). These finding strongly suggest that the efflux of copper transported by CtpA from the cytoplasm is relevant to the response to the redox stress and may be required for metalation and activity of MmcO in Mtb. [Display omitted] • CtpA is not directly required for copper detoxification in Mtb. • CtpA could deliver copper to the membrane cuproenzyme MmcO. • CtpA is required to overcome redox stress in Mtb cells. [ABSTRACT FROM AUTHOR]

Humans are predisposed to gout because they lack uricase that converts uric acid to allantoin. Rodents have uricase, resulting in low basal serum uric acid. A uricase inhibitor raises serum uric acid in rodents. There were two aims of the study in polycystic kidney disease (PKD): 1) to determine whether increasing serum uric acid with the uricase inhibitor, oxonic acid, resulted in faster cyst growth and 2) to determine whether treatment with the xanthine oxidase inhibitor, oxypurinol, reduced the cyst growth caused by oxonic acid. Orthologous models of human PKD were used: PCK rats, a polycystic kidney and hepatic disease 1 (Pkhd1) gene model of autosomal recessive PKD (ARPKD) and Pkd1RC/RC mice, a hypomorphic Pkd1 gene model. In PCK rats and Pkd1RC/RC mice, oxonic acid resulted in a significant increase in serum uric acid, kidney weight, and cyst index. Mechanisms of increased cyst growth that were investigated were proinflammatory cytokines, the inflammasome, and crystal deposition in the kidney. Oxonic acid resulted in an increase in proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice. Oxonic acid did not cause activation of the inflammasome or uric acid crystal deposition in the kidney. In Pkd1RC/RC male and female mice analyzed together, oxypurinol decreased the oxonic acid-induced increase in cyst index. In summary, increasing serum uric acid by inhibiting uricase with oxonic acid results in an increase in kidney weight and cyst index in PCK rats and Pkd1RC/RC mice. The effect is independent of inflammasome activation or crystal deposition in the kidney. NEW & NOTEWORTHY: This is the first reported study of uric acid measurements and xanthine oxidase inhibition in polycystic kidney disease (PKD) rodents. Raising serum uric acid with a uricase inhibitor resulted in increased kidney weight and cyst index in Pkd1RC/RC mice and PCK rats, elevated levels of proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice, and no uric acid crystal deposition or activation of the caspase-1 inflammasome in the kidney. [ABSTRACT FROM AUTHOR]

* Context.--Total serum bilirubin (TSB) analysis is pivotal for diagnosing neonatal hyperbilirubinemia. Because of a routine change in laboratory equipment, our TSB assay changed from a diazo to a vanadate oxidase method. Upon implementation, TSB results were substantially higher in newborns than expected based on the validation. Objective.--To investigate the application of TSB and intermethod differences in neonates and their impact on phototherapy treatment. Design.--The diazo and vanadate methods were compared directly using neonatal and adult samples. Anonymized external quality control data were analyzed to explore interlaboratory differences among 8 commercial TSB assays. Clinical patient data were extracted from the medical records to investigate the number of newborns receiving phototherapy. Results.--The mean bias of the vanadate versus the diazo TSB method was +17.4% and +3.7% in neonatal and adult samples, respectively. External quality control data showed that the bias of commercial TSB methods compared with the reference method varied from -3.6% to +20.2%. Within-method variation ranged from 5.2% to 16.0%. After implementation of the vanadate TSB method, the number of neonates treated with phototherapy increased approximately threefold. Conclusions.--Currently available TSB assays lack harmonization for the diagnosis of neonatal hyperbilirubinemia. Between-methods differences are substantially higher in neonatal compared with adult samples, highlighting the importance of including neonatal samples during assay validation. Close collaboration between laboratory specialists and clinicians is essential to prevent overtreatment or undertreatment upon the implementation of novel analyzers or assays. Also, harmonization of TSB assays, with an emphasis on neonatal application, is warranted. (Arch Pathol Lab Med. 2024;148:e40-e47; doi: 10.5858/arpa.2022-0478-OA), Neonatal jaundice is a usually benign condition with an estimated incidence of more than 60% in full-term newborns. (1) It is caused by an increase of unconjugated bilirubin, which is [...]

Cholesterol oxidases (ChOxes) are enzymes that catalyze the oxidation of cholesterol to cholest-4-en-3-one. These enzymes find wide applications across various diagnostic and industrial settings. In addition, as a pathogenic factor of several bacteria, they have significant clinical implications. The current classification system for ChOxes is based on the type of bond connecting FAD to the apoenzyme, which does not adequately illustrate the enzymatic and structural characteristics of these proteins. In this study, we have adopted an integrative approach, combining evolutionary analysis, classic enzymatic techniques and computational approaches, to elucidate the distinct features of four various ChOxes from Rhodococcus sp. (RCO), Cromobacterium sp. (CCO), Pseudomonas aeruginosa (PCO) and Burkhoderia cepacia (BCO). Comparative and evolutionary analysis of substrate-binding domain (SBD) and FAD-binding domain (FBD) helped to reveal the origin of ChOxes. We discovered that all forms of ChOxes had a common ancestor and that the structural differences evolved later during divergence. Further examination of amino acid variations revealed SBD as a more variable compared to FBD independently of FAD coupling mechanism. Revealed differences in amino acid positions turned out to be critical in determining common for ChOxes properties and those that account for the individual differences in substrate specificity. A novel look with the help of chemical descriptors on found distinct features were sufficient to attempt an alternative classification system aimed at application approach. While univocal characteristics necessary to establish such a system remain elusive, we were able to demonstrate the substrate and protein features that explain the differences in substrate profile. [Display omitted] • Current classification of cholesterol oxidases is useless for enzymatic description. • All forms of ChOxes had a common ancestor with further divergence. • Chemical descriptors turned out to be a suitable for alternative classification. • Folding-based approach can better guide the selection of enzymes for practice. [ABSTRACT FROM AUTHOR]

Background: Our earlier research revealed that the secreted lysyl oxidase-like 4 (LOXL4) that is highly elevated in triple-negative breast cancer (TNBC) acts as a catalyst to lock annexin A2 on the cell membrane surface, which accelerates invasive outgrowth of the cancer through the binding of integrin-β1 on the cell surface. However, whether this machinery is subject to the LOXL4-mediated intrusive regulation remains uncertain. Methods: Cell invasion was assessed using a transwell-based assay, protein-protein interactions by an immunoprecipitation-Western blotting technique and immunocytochemistry, and plasmin activity in the cell membrane by gelatin zymography. Results: We revealed that cell surface annexin A2 acts as a receptor of plasminogen via interaction with S100A10, a key cell surface annexin A2-binding factor, and S100A11. We found that the cell surface annexin A2/ S100A11 complex leads to mature active plasmin from bound plasminogen, which actively stimulates gelatin digestion, followed by increased invasion. Conclusion: We have refined our understanding of the role of LOXL4 in TNBC cell invasion: namely, LOXL4 mediates the upregulation of annexin A2 at the cell surface, the upregulated annexin 2 binds S100A11 and S100A10, and the resulting annexin A2/S100A11 complex acts as a receptor of plasminogen, readily converting it into active-form plasmin and thereby enhancing invasion. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) produced by NADPH oxidases (NOX, a key source of ROS in vascular cells) are involved in the regulation of vascular tone, but this has been explored mainly for adult organisms. Importantly, the mechanisms of vascular tone regulation differ significantly in early postnatal ontogenesis and adulthood, while the vasomotor role of ROS in immature systemic arteries is poorly understood. We tested the hypothesis that the functional contribution of NADPH oxidase-derived ROS to the regulation of peripheral arterial tone is higher in the early postnatal period than in adulthood. We studied saphenous arteries from 10‐ to 15‐day‐old ("young") and 3‐ to 4-month-old ("adult") male rats using lucigenin-enhanced chemiluminescence, quantitative PCR, Western blotting, and isometric myography. We demonstrated that both basal and NADPH-stimulated superoxide anion radical (O 2 •−) production was significantly higher in the arteries from young in comparison to adult rats. Importantly, pan-inhibitor of NADPH oxidase VAS2870 (10 μM) reduced NADPH-induced O 2 •− production in arteries of young rats. Saphenous arteries of both young and adult rats demonstrated high levels of Nox2 and Nox4 mRNAs, while Nox1 and Nox3 mRNAs were not detected. The protein contents of NOX2 and NOX4 were significantly higher in arterial tissue of young compared to adult animals. Moreover, VAS2870 (10 μM) had no effect on methoxamine-induced contractile responses of adult arteries but decreased them significantly in young arteries; such effect of VAS2870 persisted after removal of the endothelium. Finally, NOX2 inhibitor GSK2795039 (10 μM), but not NOX1/4 inhibitor GKT137831 (10 μM) weakened methoxamine-induced contractile responses of arteries from young rats. Thus, ROS produced by NOX2 have a pronounced contractile influence in saphenous artery smooth muscle cells of young, but not adult rats, which is associated with the increased vascular content of NOX2 protein at this age. [Display omitted] • O 2.•− production is higher in saphenous artery of young compared to adult rats • NADPH-oxidases are important source of O 2.•− in saphenous artery of young rats • NOX2 and NOX4 contents are higher in arteries of young compared to adult rats. • O 2.•− produced by NOX2 promotes arterial contraction in young, but not adult rats [ABSTRACT FROM AUTHOR]

Flavoenzymes can mediate a large variety of oxidation reactions through the activation of oxygen. However, the O2 activation chemistry of flavin enzymes is not yet fully exploited. Normally, the O2 activation occurs at the C4a site of the flavin cofactor, yielding the flavin C4a‐(hydro)hydroperoxyl species in monooxygenases or oxidases. Using extensive MD simulations, QM/MM calculations and QM calculations, our studies reveal the formation of the common nucleophilic species, Flavin‐N5OOH, in two distinct flavoenzymes (RutA and EncM). Our studies show that Flavin‐N5OOH acts as a powerful nucleophile that promotes C−N cleavage of uracil in RutA, and a powerful base in the deprotonation of substrates in EncM. We reason that Flavin‐N5OOH can be a common reactive species in the superfamily of flavoenzymes, which accomplish generally selective general base catalysis and C−X (X=N, S, Cl, O) cleavage reactions that are otherwise challenging with solvated hydroxide ion base. These results expand our understanding of the chemistry and catalysis of flavoenzymes. [ABSTRACT FROM AUTHOR]

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications. [ABSTRACT FROM AUTHOR]

Green waste (GW) is generated by the maintenance of public or private green spaces. It is necessary to find ecological alternatives for GW utilization, aiming to avoid accumulation of this material at the environment. In this research, the production of laccase (Lac), lignin peroxidase (LiP), manganese peroxidase (MnP) and unspecific peroxygenase (UnP) produced by Trametes versicolor grown on GW as a substrate and on polyurethane foam (PUF) as an inert support in solid state fermentation was evaluated. T. versicolor showed higher values of Lac, MnP, UnP and LiP activities (34, 943, 1023 and 766 U/gS, respectively) when grown on GW than when grown on PUF (10.9, 588, 559 and 229 U/gS, respectively). These results suggest that T. versicolor produced Lac inducible and constitutively, while LiP, MnP and UnP were induced by GW at the beginning of fungal growth, however, these enzymes were constitutive and inducible during the rest of the fermentation. The production of oxidases and peroxidases was induced and increased by GW. It is suggested that LiP is involved (as a constitutive enzyme) at the beginning of the exponential phase, while MnP and UnP participate in fungal growth at the end of fermentation. To our knowledge, this is the first detailed study on the main lignincellulosedegrading fungal enzymes involved in GW degradation by fungi. In particular, the relevance of UnP was showed as peroxidase involved in lignocellulosic substrates biodegradation. [ABSTRACT FROM AUTHOR]

Plastid terminal oxidases (PTOXs) are essential for maintaining photosynthetic efficiency and cellular redox homeostasis. Astaxanthin, a carotenoid pigment with antioxidant properties, is synthesized and accumulates in response to oxidative stress induced by high-light intensity or nutrient limitation. It suggests that PTOX may impact astaxanthin biosynthesis under environmental stress conditions due to its involvement in ROS regulation. The ptox1 gene is thought to have a conserved role in safeguarding the photosynthetic apparatus from over-reduction and participating in energy dissipation. On the other hand, the ptox2 gene seems to be involved in the evolution of astaxanthin synthesis and adaptive responses to diverse environmental stressors. Efficient gene silencing strains were developed in Chlamydomonas reinhardtii CC849 for ptox1 and ptox2. The study found that the ptox2 gene correlates highly with resistance to intense light stress. Furthermore, the ptox2 gene showed increased activity under high salt stress conditions, indicating its importance in stress coping mechanisms. The quantification of astaxanthin in the gene-silenced strains revealed that ptox1 acts as a positive regulator, while ptox2 functions as a negative regulator of astaxanthin accumulation. Understanding the coordination between ptox1 and ptox2 could clarify the synergistic actions of these genes in maintaining photosynthetic performance and redox balance under fluctuating environmental conditions. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes, which causes human listeriosis after consumption of contaminated food, can adapt and survive under a wide range of physiological and chemical stresses. In this study, the overall proteomic response of the L. monocytogenes strain F2365—a strain with mutations limiting its ability to tolerate acidic conditions—to progressive non-thermal acidic inactivation was investigated. The challenge process was investigated in the early stationary growth phase where F2365 cultures were acidified (pH 3.0, HCl) at 5 min, 1 h, and 2 h, generating pH 4.8, pH 4.1, and pH 3.5, respectively, with protein abundance measured using iTRAQ. Approximately 73 proteins increased in abundance and 8 declined when acidic stress became non-growth-permissive (pH < 4.1) and inactivation accelerated to approximately 2 log units/h. The functional categories of responding proteins were broad but the proteins involved were specific in nature and did not include whole pathways. Many responses likely accentuate energy conservation and compensate vital metabolic processes. For example, further repression of FlaA, normally repressed under acidic stress, occurs accompanied by an increase in quinol oxidase subunit QoxA and glycerol kinase GlpK. Proteins maintaining cell wall integrity, such as Iap and CwlO, manifested the overall largest abundance increase trend. Virulence proteins were also induced, including InlA, InlC, Hyl, Mpl, PlcA, and PlcB, suggesting that acidification may have mimicked conditions inducing some host survival traits. The overall suite of proteins affected appears to be the “last ditch” responses to non-thermal inactivation above and beyond the standard protections afforded in the stationary-growth phase. The array of proteins found here may provide a deeper understanding of the physiological responses of this pathogen during non-thermal inactivation. [ABSTRACT FROM AUTHOR]

Background: Oxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily. Results: In this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold. Conclusions: The phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions. [ABSTRACT FROM AUTHOR]

To conveniently monitor bioactive cysteine (Cys) and Fe2+ in practice, a kind of poly-β-cyclodextrin strengthen praseodymium oxide (Pr6O11) porous oxidase mimic (p-β-CD@Pr6O11) was constructed by virtue of the strong coordination between nano Pr6O11 and poly-β-cyclodextrin substrate. After its microstructure and physicochemical property were characterized in detail, it was noted that porous p-β-CD@Pr6O11 exhibited excellent enzyme-like catalytic activity to accelerate the oxidation of 3,3′,5,5,′-tetramethylbanzidine (TMB) and 2,2′-azinobis (3-ethylbenzo-thiazoline-6-sulfonic acid) ammonium salt (ABTS) with significant color-enhancement effect in the air. Based on the signal amplification, trace Cys could exclusively deteriorate the UV-vis absorbance at 653 nm of p-β-CD@Pr6O11-TMB and Fe2+ alter the one at 729 nm of p-β-CD@Pr6O11-ABTS with visual color changes. Under the optimized conditions, the proposed p-β-CD@Pr6O11-TMB and p-β-CD@Pr6O11-ABTS systems were successfully applied for dual-channel monitoring of Cys in Cys capsules and fetal bovine serum and Fe2+ in agricultural products with quite low detection limits, i.e., 7.8×10−9 mol·L−1 for Cys and 6.93×10−8 mol·L−1 (S/N=3) for Fe2+, respectively. The synergetic-enhancement detection mechanisms to Cys and Fe2+ were also proposed. [ABSTRACT FROM AUTHOR]

Nature utilizes three distinct pathways to synthesize the essential enzyme cofactor heme. The coproporphyrin III-dependent pathway, predominantly present in Bacillaceae, employs an oxygen-dependent coproporphyrinogen III oxidase (CgoX) that converts coproporphyrinogen III into coproporphyrin III. In this study, we report the bioinformatic-based identification of a gene called ytpQ, encoding a putative oxygen-independent counterpart, which we propose to term CgoN, from Priestia (Bacillus) megaterium. The recombinantly produced, purified, and monomeric YtpQ (CgoN) protein is shown to catalyze the oxygenindependent conversion of coproporphyrinogen III into coproporphyrin III. Minimal non-enzymatic conversion of coproporphyrinogen III was observed under the anaerobic test conditions employed in this study. FAD was identified as a cofactor, and menadione served as an artificial acceptor for the six abstracted electrons, with a KM value of 3.95 μmol/L and a kcat of 0.63 per min for the substrate. The resulting coproporphyrin III, in turn, acts as an effective substrate for the subsequent enzyme of the pathway, the coproporphyrin III ferrochelatase (CpfC). Under aerobic conditions, oxygen directly serves as an electron acceptor, but is replaced by the more efficient action of menadione. An AlphaFold2 model of the enzyme suggests that YtpQ adopts a compact triangular shape consisting of three domains. The N-terminal domain appears to be flexible with respect to the rest of the structure, potentially creating a ligand binding site that opens and closes during the catalytic cycle. A catalytic mechanism similar to the oxygen-independent protoporphyrinogen IX oxidase PgoH1 (HemG), based on the flavin-dependent abstraction of six electrons from coproporphyrinogen III and their potential quinone-dependent transfer to a membrane-localized electron transport chain, is proposed. [ABSTRACT FROM AUTHOR]

To conveniently monitor bioactive cysteine (Cys) and Fe2+ in practice, a kind of poly-β-cyclodextrin strengthen praseodymium oxide (Pr6O11) porous oxidase mimic (p-β-CD@Pr6O11) was constructed by virtue of the strong coordination between nano Pr6O11 and poly-β-cyclodextrin substrate. After its microstructure and physicochemical property were characterized in detail, it was noted that porous p-β-CD@Pr6O11 exhibited excellent enzyme-like catalytic activity to accelerate the oxidation of 3,3′,5,5,′-tetramethylbanzidine (TMB) and 2,2′-azinobis (3-ethylbenzo-thiazoline-6-sulfonic acid) ammonium salt (ABTS) with significant color-enhancement effect in the air. Based on the signal amplification, trace Cys could exclusively deteriorate the UV-vis absorbance at 653 nm of p-β-CD@Pr6O11-TMB and Fe2+ alter the one at 729 nm of p-β-CD@Pr6O11-ABTS with visual color changes. Under the optimized conditions, the proposed p-β-CD@Pr6O11-TMB and p-β-CD@Pr6O11-ABTS systems were successfully applied for dual-channel monitoring of Cys in Cys capsules and fetal bovine serum and Fe2+ in agricultural products with quite low detection limits, i.e., 7.8×10−9 mol·L−1 for Cys and 6.93×10−8 mol·L−1 (S/N=3) for Fe2+, respectively. The synergetic-enhancement detection mechanisms to Cys and Fe2+ were also proposed. [ABSTRACT FROM AUTHOR]

Laccases are multicopper oxidases able to oxidize several phenolic compounds and find application in numerous industrial applications. Among laccase producers, white-rot fungi represent a valuable source of multiple isoforms and isoenzymes of these multicopper oxidases. Here we describe the identification, biochemical characterization, and application of laccase 2 from Trametes polyzona (TP-Lac2), a basidiomycete fungus emerged among others that have been screened by plate assay. This enzyme has an optimal temperature of 50 °C and in acidic conditions it is able to oxidize both phenolic and non-phenolic compounds. The ability of TP-Lac2 to decolorize textile dyes was tested in the presence of natural and synthetic mediators at 30 °C and 50 °C. Our results indicate that TP-Lac2 most efficiently decolorizes (decolorization rate > 75%) malachite green oxalate, orange G, amido black10B and bromocresol purple in the presence of acetosyringone and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonate)—ABTS. Overall, the laccase mediator system consisting of TP-Lac2 and the natural mediator acetosyringone has potential as an environmentally friendly alternative for wastewater treatment in the textile industry. Key points: A novel laccase from T. polyzona was induced by the addition of veratryl alcohol TP-Lac2 was heterologous expressed in S. cerevisiae and biochemically characterized TP-Lac2 efficiently decolorizes textile dyes using acetosyringone as mediator [ABSTRACT FROM AUTHOR]

Polychlorinated biphenyls (PCBs) are persistent organic pollutants in the environment that are responsible for many adverse health effects. Bioremediation appears to be a healthy and cost-effective alternative for remediating PCB-contaminated environments. While some microbial species have been observed to be capable of transforming PCBs, only two different microbial pathways (rdh and bph pathways) have been described to be involved in PCB transformations. Ligninolytic enzymes have been observed or are under suspicion in some microbial PCB transformations. However, the role of these promising PCB-transforming enzymes, which are produced by fungi and some aerobic bacteria, is still unclear. The present review describes their role by identifying microbial PCB-transforming species and their reported ligninolytic enzymes whether proven or suspected to be involved in PCB transformations. There are several lines of evidence that ligninolytic enzymes are responsible for PCB transformations such as (1) the ability of purified laccases from Myceliophthora thermophila, Pycnoporus cinnabarinus, Trametes versicolor, Cladosporium sp, and Coprinus cumatus to transform hydroxy-PCBs; (2) the increased production of laccases and peroxidases by many fungi in the presence of PCBs; and (3) the enhanced PCB transformation by Pseudomonas stutzeri and Sinorhizobium meliloti NM after the addition of ligninolytic enzyme enhancers. However, if the involvement of ligninolytic enzymes in PCB transformation is clearly demonstrated in some fungal species, it does not seem to be implicated in all microbial species suggesting other still unknown metabolic pathways involved in PCB transformation and different from the bph and rdh pathways. Therefore, PCB transformation may involve several metabolic pathways, some involving ligninolytic enzymes, bph or rdh genes, and some still unknown, depending on the microbial species. In addition, current knowledge does not fully clarify the role of ligninolytic enzymes in PCB oxidation and dechlorination. Therefore, further studies focusing on purified ligninolytic enzymes are needed to clearly elucidate their role in PCB transformation. [ABSTRACT FROM AUTHOR]

Monoamine oxidases (MAOs) are pivotal regulators of neurotransmitters in mammals, while microbial MAOs have been shown to be valuable biocatalysts for enantioselective synthesis of pharmaceutical compounds or precursors thereof. To extend the knowledge of how MAOs function at the molecular level and in order to provide more biocatalytic tools, we set out to identify and study a robust bacterial variant: a MAO from the thermophile Thermoanaerobacterales bacterium (MAOTb). MAOTb is highly thermostable with melting temperatures above 73 °C and is well expressed in Escherichia coli. Substrate screening revealed that the oxidase is most efficient with n-alkylamines with n-heptylamine being the best substrate. Presteady-state kinetic analysis shows that reduced MAOTb rapidly reacts with molecular oxygen, confirming that it is a bona fide oxidase. The crystal structure of MAOTb was resolved at 1.5 °A and showed an exceptionally high similarity with the two human MAOs, MAO A and MAO B. The active site of MAOTb resembles mostly the architecture of human MAO A, including the cysteinyl protein-FAD linkage. Yet, the bacterial MAO lacks a C-terminal extension found in human MAOs, which explains why it is expressed and purified as a soluble protein, while the mammalian counterparts are anchored to the membrane through an a-helix. MAOTb also displays a slightly different active site access tunnel, which may explain the specificity toward long aliphatic amines. Being an easy-to-express, thermostable enzyme, for which a high-resolution structure was elucidated, this bacterial MAO may develop into a valuable biocatalyst for synthetic chemistry or biosensing. [ABSTRACT FROM AUTHOR]

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia–reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR. [ABSTRACT FROM AUTHOR]