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Cancer cells exhibit higher proliferation rates than normal cells, and as a consequence, a higher nutritional demand for metabolites such as amino acids. Such cells demonstrate high expression of amino acid transporters and are significantly dependent on the external uptake of amino acids. Moreover, some types of cancer cells exhibit oncogenic mutations that render them auxotrophic to certain amino acids. This metabolic difference between tumor and normal cells has been explored for developing anticancer drugs. Enzymes capable of depleting certain amino acids in the bloodstream can be employed to inhibit the proliferation of cancer cells and promote cell death. Certain microbial enzymes, such as l -asparaginase and l -amino acid oxidases, have been studied for this purpose. In this paper, we discuss the role of l -asparaginase, the only enzyme currently used as a chemotherapeutic agent. We also review the studies on a new potential antineoplastic agent, l -lysine α-oxidase, an enzyme of l -amino acid oxidase family.

The cytochrome bc1-aa3 supercomplex plays an essential role in the cellular respiratory system of Mycobacterium Tuberculosis. It transfers electrons from menaquinol to cytochrome aa3 (Complex IV) via cytochrome bc1 (Complex III), which reduces the oxygen. The electron transfer from a variety of donors into oxygen through the respiratory electron transport chain is essential to pump protons across the membrane creating an electrochemical transmembrane gradient (proton motive force, PMF) that regulates the synthesis of ATP via the oxidative phosphorylation process. Cytochrome bc1-aa3 supercomplex in M. tuberculosis is, therefore, a major drug target for antibiotic action. In recent years, several respiratory chain components have been targeted for developing new candidate drugs, illustrating the therapeutic potential of obstructing energy conversion of M. tuberculosis. The recently available cryo-EM structure of mycobacterial cytochrome bc1-aa3 supercomplex with open and closed conformations has opened new avenues for understanding its structure and function for developing more effective, new therapeutics against pulmonary tuberculosis. In this review, we discuss the role and function of several components, subunits, and drug targeting elements of the supercomplex cytochrome bc1-aa3 and its potential inhibitors in detail.

Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) is a significant agricultural crop pest in Indonesia, causing significant economic losses in recent years. This species' ability to survive on a wide variety of host plants provides an adaptive advantage for survival in the environment, which is facilitated by its high mobility, fecundity, and capability to acquire resistance to a broad spectrum of chemical pesticides. It is well-established that knowledge of diversity and evolutionary origins facilitate the development of disease management strategies. In the present study, we report the morphology, diversity and phylogeny analysis of S. exigua from North Sulawesi, Indonesia. The specimen from Rurukan have a body size and other segments that are longer than in Langowan and Modoinding. Dendrogram analysis shows that the similarity distance based on morphology ranges from 1-25%, which forms four clusters, where the specimen from Rurukan is separated from the rest of the specimens. The phylogeny of S. exigua from North Sulawesi, Indonesia, based on CO1 (Cytochrome c oxidase subunit 1) gene fragment, which is juxtaposed with CO1 data of the allied species from many geographical locations. A total of twenty-five isolates representing Indonesia, Japan, Germany, Thailand, India, UK, USA, Spain and Australia were compared. Nineteen sequences of S. exigua retrieved from GenBank were selected as references based on previous published phylogenic trees. The twenty-four isolates were scattered in two distinct clades indicating S. exigua is polyphyletic, but S. exigua from North Sulawesi, Indonesia is monophyletic. [ J Adv Biotechnol Exp Ther 2022; 5(1.000): 136-147]

To clarify the precise subunit composition of the respiratory supercomplex of Corynebacterium glutamicum, several wash conditions were examined. MEGA (9 + 10) wash-buffer (0.5%) was used for this purpose and two-step column chromatography was performed. Almost equal amounts of cytochrome c, b, and a were observed in the purified fraction, estimated by their different absorption spectra. The 833 kDa and 685 kDa bands were observed in the clear native polyacrylamide gel electrophoresis (CN-PAGE) of the purified fraction. Both bands were stained using N,N',N′,N-tetramethyl-p-phenylenediamine (TMPD) oxidase dye, and the 833 kDa band was also stained using NADH oxidase dye. The 3D map reconstructed from the 833 kDa band indicated that the bcc complex and aa3 oxidase are heterodimers. Lastly, electron transfer from NADH to the bcc-aa3 supercomplex was observed. The 833 kDa band is the supercomplex, which includes the heterodimer cytochrome bcc complex and cytochrome aa3 oxidase, as well as the monomer NDH-II. Hence, we termed the 833 kDa band the extended supercomplex (ESC).

Aims: We aimed to explore the role of nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (Nox4) in the regulation of hypercalciuria-induced renal oxidative damage and crystal depositions...

Chemodynamic therapy (CDT) is widely explored for tumor-specific therapy by converting endogenous H2O2 to lethal ·OH to destroy cancer cells. However, ·OH scavenging by glutathione (GSH) and insufficient intratumoral H2O2 levels seriously hinder the application of CDT. Herein, we reported the fabrication of copper ion-doped ZIF-8 loaded with gold nanozymes and doxorubicin hydrochloride (DOX) for the chemotherapy and CDT synergistic treatment of tumors with the assistance of tumor microenvironment (TME)-activated fluorescence imaging. The Cu2+-doped ZIF-8 shell was gradually degraded to release DOX and gold nanoclusters responding to the acidic TME. The fluorescence signal of the tumor region was acquired after the quenched fluorescence of the gold nanoclusters by Cu2+ and DOX by aggregation-induced quenching was turned on because of the interaction of GSH with Cu2+ and the release of free DOX. The Cu2+ ions could deplete the GSH via redox reactions and the generated Cu+ could convert internal H2O2 to ·OH for tumor CDT. The chemotherapeutic effect of DOX was strengthened through drug efflux inhibition and drug sensitivity increase due to the consumption of GSH and ·OH burst. Moreover, DOX could raise the level of H2O2 and augment the effect of CDT. In addition, the fluorescent gold nanoclusters not only served as a peroxidase to convert H2O2 to ·OH but also employed as an oxidase to consume GSH, resulting in the amplification of chemotherapy and CDT. This work presents an approach to construct tumor microenvironment-activated theranostic probes without external stimuli and to achieve the tumor elimination through cascade reactions and synergistic treatment.

Jatropha curcas received a lot of attention as a biodiesel feedstock in several countries around the world. The leaf area (LA) estimation is important for biological research due to its close relationship to soil fertility, plant physiological parameters, and photosynthetic efficiency. Allometric models for reliable and accurate models for estimating the leaf area of Jatropha curcas plants based on non-destructive measurements of leaf length (L), width (W), and/ or leaf margins length (P) were developed. Jatropha plant leaves were randomly selected for model construction to estimate the leaf area using L, W, and P leaf values. Simple and accurate equations were obtained, based on leaf width (AL = W1.795), R2 = 0.987, rib length (AL = L32.081), R2 = 0.989, width and rib length (AL = LW0.320), R2 = 0.997, rib length and leaf periphery segment (AL = LP1P120.274), R2 = 0.995, and half rib and width length (AL = LW/0.50.467), R2 = 0.994. The equations were accurate for estimating leaf area of small, medium, and large size of Jatropha leaves.

Ralstonia solanacearum RSc0454 is predicted as a FAD-linked oxidase based on protein homologies, while it contains distinct domains of lactate dehydrogenase and succinate dehydrogenase. A previous study demonstrated that RSc0454 exhibits lactate dehydrogenase activity using pyruvate and NADH as substrates, and is essential for pathogenicity of R. solanacearum. Here, we genetically characterized involvement of RSc0454 on bacterial growth and expression of genes for the type III secretion system (T3SS, a pathogenicity determinant) in R. solanacearum. The RSc0454 mutant grew normally in rich medium but grew faintly in host plants, and failed to grow in minimal medium. Supplementary succinate but not lactate could substantially restore some phenotypes of RSc0454 mutants, including faint growth in host plants, diminished growth in the minimal medium, and lost pathogenicity toward host plants. Expression of T3SS genes is directly controlled by a master regulator, HrpB, and hrpB expression is positively regulated by HrpG and PrhG in parallel ways. Deletion of RSc0454 substantially reduced expression levels of hrpB and T3SS both in vitro and in planta. Moreover, RSc0454 is revealed to be required for the T3SS expression via HrpG and PrhG, although through some novel pathway, and impaired expression of these genes was not due to growth deficiency of RSc0454 mutants. RSc0454 is suggested to be important for redox balance inside cells, and supplementary NADH partially restored diminished growth of the RSc0454 mutant in the minimal medium only in the presence of succinate at some moderate concentrations, indicating that the unbalanced redox in the RSc0454 mutant might be responsible for its diminished growth in the minimal medium. Taken together, these results provide novel insights into the understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on expression of the T3SS in R. solanacearum. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Microglial activation and neuroinflammation induced by amyloid β (Aβ) play pivotal roles in Alzheimer's disease (AD) pathogenesis. Astragaloside IV (AS-IV) is one of the major active compounds of the traditional Chinese medicine Astmgali Radix. It has been reported that AS-IV could protect against Aβ-induced neuroinflammation and cognitive impairment, but the underlying mechanisms need to be further clarified. In this study, the therapeutic effects of AS-IV were investigated in an oligomeric Aβ (oAβ) induced AD mice model. The effects of AS-IV on microglial activation, neuronal damage and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were further studied. Different doses of AS-IV were administered intragastrically once a day after intracerebroventricularly oAβ injection. Results of behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test revealed that AS-IV administration could significantly ameliorate oAβ-induced cognitive impairment in a dose dependent manner. Enzyme linked immunosorbent assay (ELISA) results showed that increased levels of reactive oxygen species (ROS), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and IL-6 in hippocampal tissues induced by oAβ injection were remarkably inhibited after AS-IV treatment. OAβ induced microglial activation and neuronal damage was significantly suppressed in AS-IV-treated mice brain, observed in immunohistochemistry results. Furthermore, oAβ upregulated protein expression of NADPH oxidase subunits gp91phox, p47phox, p22phox and p67phox were remarkably reduced by AS-IV in Western blotting assay. These results revealed that AS-IV could ameliorate oAβ-induced cognitive impairment, neuroinflammation and neuronal damage, which were possibly mediated by inhibition of microglial activation and down-regulation of NADPH oxidase protein expression. Our findings provide new insights of AS-IV for the treatment of neuroinflammation related diseases such as AD.

Palm oil has long been incriminated in obesity, and this obesity would be responsible for the development of cardiac fibrosis, several authors have evoked the role of free radicals in the pathophysiological mechanism of the fibrotic response linked to obesity; the aim of this study was therefore to evaluate the profile of NOX2 in the development of cardiac fibrosis in rats subjected to a diet rich in the fat of several vegetable oils, in this case palm oil. A total of forty young male Wistar rats were subjected to several diets (soybean, red and branched palm oil, olive and lard). After twelve weeks of experimentation, the rats were sacrificed after anaesthesia, and the parameters of oxidative stress, inflammation and the level of interstitial fibrosis of the heart were assessed. Our study showed that red palm oil consumption did not lead to overexpression of oxidative stress parameters and inflammatory RNA markers. The expression of myocardial nicotine adenine dinucleotide phosphate oxidase did not change in rats consuming red palm oil compared to the control diet. However, consumption of palm olein, olive and lard resulted in a significant change in myocardial nicotine adenine dinucleotide phosphate oxidase activity. This study seems to show that red palm oil, because of its richness in antioxidants, would be less deleterious for the heart. Key words: Oxidative stress-inflammation-palm oil-cardiac fibrosis.

In this paper, the application of biological methods to reduce biogenic amine content in foods was introduced. Biogenic amine oxidase, a special protein that can degrade biogenic amine into acetaldehyde, hydrogen peroxide and ammonia, has been introduced in this paper, and two major amine oxidases and their degradation effects on different biogenic amines were briefly reviewed. In addition, various microorganisms that could produce amine oxidase were summarized in this paper, and their application in the fermentation was shown. This short review summarizes the important biological methods currently used to degrade biogenic amines and provides new theoretical guidance for removing or reducing the biogenic amines in foods.

Significance: The significant role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in signal transduction is mediated by the production of reactive oxygen species (ROS)...

The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.

Decaprenylphosphoryl-β-D-ribose oxidase (DprE1) plays important roles in the biosynthesis of mycobacterium cell wall. DprE1 inhibitors have shown great potentials in the development of new regimens for tuberculosis (TB) treatment. In this study, an integrated molecular modeling strategy, which combined computational bioactivity fingerprints and structure-based virtual screening, was employed to identify potential DprE1 inhibitors. Two lead compounds (B2 and H3) that could inhibit DprE1 and thus kill Mycobacterium smegmatis in vitro were identified. Moreover, compound H3 showed potent inhibitory activity against Mycobacterium tuberculosis in vitro (MIC(Mtb) = 1.25 μM) and low cytotoxicity against mouse embryo fibroblast NIH-3T3 cells. Our research provided an effective strategy to discover novel anti-TB lead compounds.

A suboptimal blood vitamin C (ascorbate) level increases the risk of several chronic diseases. However, the detection of hypovitaminosis C is not a simple task, as ascorbate is unstable in blood samples. In this study, we examined the serum proteome of mice lacking the gulonolactone oxidase (Gulo) required for the ascorbate biosynthesis. Gulo-/- mice were supplemented with different concentrations of ascorbate in drinking water, and serum was collected to identify proteins correlating with serum ascorbate levels using an unbiased label-free liquid chromatography-tandem mass spectrometry global quantitative proteomic approach. Parallel reaction monitoring was performed to validate the correlations. We uncovered that the serum proteome profiles differ significantly between male and female mice. Also, unlike Gulo-/- males, a four-week ascorbate treatment did not entirely re-establish the serum proteome profile of ascorbate-deficient Gulo-/- females to the optimal profile exhibited by Gulo-/- females that never experienced an ascorbate deficiency. Finally, the serum proteins involved in retinoid metabolism, cholesterol, and lipid transport were similarly affected by ascorbate levels in males and females. In contrast, the proteins regulating serum peptidases and the protein of the acute phase response were different between males and females. These proteins are potential biomarkers correlating with blood ascorbate levels and require further study in standard clinical settings. The complete proteomics data set generated in this study has been deposited to the public repository ProteomeXchange with the data set identifier: PXD027019.

Sustainable wheat production in drought prone areas can be achieved by developing resilient wheat varieties. In the present study, chemical mutagenesis was used to induce mutations in a cultivated wheat variety ‘NN-Gandum-1’. In total, 44 mutants were selected based on their high yield potential for exposing to well-watered (W 1) and rainfed (W 2) conditions for one season. Then, 24 mutants were selected and were exposed to W 1 and W 2 regimes. On the basis of least relative reduction in physiological parameters under W 2 regime, five mutants were selected for conducting exome capturing assays. In total, 184 SNPs were identified in nine genes (ABC transporter type 1, aspartic peptidase, cytochrome P450, transmembrane domain, heavy metal-associated domain, HMA, NAC domain, NAD (P)-binding domain, S-type anion channel, Ubiquitin-conjugating enzyme E2 and UDP-glucuronosyl/UDP-glucosyltransferase). Maximum number of mutations were observed in chr.2D, which contained mutations in three genes, i.e. ABC transporter type 1, NAD (P)-binding domain and UDP-glucuronosyl/UDP-glucosyltransferase which may have a role in conferring drought tolerance. The selected mutants were further tested for studying their biochemical responses under both the regimes for 2 years. The extent of membrane damage was estimated through malondialdehyde and hydrogen per oxidase, and tolerance to drought stress was assessed via antioxidant enzymes in leaves. The selected mutants under drought stress increased the accumulation of proline content, total soluble sugars, total free amino acids, while decreased total chlorophyll content, carotenoids and total soluble protein. These mutants can further be explored to understand the genetic circuits of drought tolerance in wheat.

In modern agricultural practice, heavy metal (HM) contamination is one of the main abiotic stress threatening sustainable agriculture, crop productivity, and disturb natural soil microbiota. Different reclamation techniques are used to restore the contaminated site; however, they are either costly or unable to remove contaminant when concentration is very low. In such circumstances, bioremediation is used as a novel technique involving microbes for soil restoration. In the current project, Aspergillus welwitschiae(Bk) efficiently endure metal stress (i.e., Cr-VI and As-V in the form of K2Cr2O7 and Na3AsO4) up to 1200 μg/mL and enhanced the production of phytohormones, i.e., 54.83 μg/mL of indole acetic acid (IAA) compared to control 15.56 μg/mL, solubilized inorganic phosphate, and produced stress-related metabolites. The isolate Bk was able to enhance growth of soybean by showing higher root shoot length and fresh/dry weight under stress (p

A number of approaches have been developed over the years to manage cancer, such as chemotherapy using low-molecular-mass molecules and radiotherapy. Here, enzymes can also find useful applications. Among them, oxidases have attracted attention because of their ability to produce reactive oxygen species (ROS, especially hydrogen peroxide) in tumors and potentially modulate the production of this cytotoxic compound when enzymes active on substrates present in low amounts are used, such as the d-amino acid oxidase and d-amino acid couple system. These treatments have been also developed for additional cancer treatment approaches, such as phototherapy, nutrient starvation, and metal-induced hydroxyl radical production. In addition, to improve tumor specificity and decrease undesired side effects, oxidases have been targeted by means of nanotechnologies and protein engineering (i.e., by designing chimeric proteins able to accumulate in the tumor). The most recent advances obtained by using six different oxidases (i.e., the FAD-containing enzymes glucose oxidase, d- and l-amino acid oxidases, cholesterol oxidase and xanthine oxidase, and the copper-containing amine oxidase) have been reported. Anticancer therapy based on oxidase-based ROS production has now reached maturity and can be applied in the clinic.

Cytochrome P450 1A1 (CYP1A1) is a member of a subfamily of enzymes involved in the metabolism of both endogenous and exogenous substrates and the chemical activation of xenobiotics to carcinogenic derivatives. Here, the effects of nicotine, a major psychoactive compound present in cigarette smoke, on CYP1A1 expression and human hepatocellular carcinoma (HepG2) cell proliferation were investigated. Nicotine stimulated CYP1A1 expression via the transcription factors, activator protein 1, nuclear factor-kappa B, and the aryl hydrocarbon receptor (AhR) signaling pathway. Pharmacological inhibition and mutagenesis studies indicated that p38 mitogen-activated protein kinase, as well as RelA (or p65), mediated the upregulation of CYP1A1 of nicotine in HepG2 cells. The antioxidant compound, N-acetyl-cysteine, abrogated nicotine-activated production of reactive oxygen species and inhibited CYP1A1 expression by nicotine. Furthermore, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was inhibited by diphenyleneiodonium (an NADPH oxidase inhibitor). Thus, these results demonstrated that AhR played an important role in nicotine-induced CYP1A1 expression. Additionally, liver hepatocellular carcinoma HepG2 cells treated with nicotine exhibited markedly enhanced proliferation via CYP1A1 expression and Akt activation.

Background: D-amino acid oxidase (DAO) is an H2O2-generating enzyme, and tumor growth suppression by selective delivery of porcine DAO in tumors via the cytotoxic action of H2O2 has been reported. DAO isolated from Fusarium spp. (fDAO) shows much higher enzyme activity than porcine DAO, although the application of fDAO for antitumor treatment has not yet been determined. Objective: The purpose of this study was to prepare enzymatically highly active pegylated-fDAO, and to determine whether it accumulates in tumors and exerts a potent antitumor effect in tumor-bearing mice. Methods: Polyethylene glycol (PEG; Mw. 2000) was conjugated to fDAO to form PEGylated fDAO (PEG-fDAO). PEGfDAO was intravenously administered into S180 tumor-bearing mice, and the body distribution and antitumor activity of PEG-fDAO was determined. Results: The enzyme activity of PEG-fDAO was 26.1 U/mg, which was comparable to that of fDAO. Intravenously administered PEG-fDAO accumulated in tumors with less distribution in normal tissue except in the plasma. Enzyme activity in the tumor was 60-120 mU/g-tissue over 7-20 h after i.v. injection of 0.1 mg of PEG-fDAO. To generate the H2O2 in the tumor tissue, PEG-fDAO was intravenously administered, and then, D-phenylalanine was intraperitoneally administered after a lag time. No remarkable tumor suppression effect was observed under conditions used in this study, compared to the non-treated group. Conclusion: The results suggest that PEG-fDAO maintained high enzymatic activity after pegylation. Treatment with PEGfDAO conferred high enzyme activity on tumor tissue; 3–6 fold higher than that of previously reported pDAO; however, high enzyme activity in the plasma limited repeated treatment owing to lethal toxicity, which seemingly led to poor therapeutic outcome. Overall, the use of PEG-fDAO is promising for antitumor therapy, although the suppression of DAO activity in the plasma would also be required rather than only the increase in DAO activity in the tumor for an antitumor effect.

Some rare sugars can be potently medicinal, such as l -gulose. In this study, we present a novel alditol oxidase (fAldOx) from the soil fungus Penicillium sp. KU–1, and its application for the effective production of l -gulose. To the best of our knowledge, this is the first report of a successful direct conversion of d -sorbitol to l -gulose. We further purified it to homogeneity with a ∼108-fold purification and an overall yield of 3.26%, and also determined the effectors of fAldOx. The enzyme possessed broad substrate specificity for alditols such as erythritol (kcat/KM, 355 m −1 s−1), thus implying that the effective production of multiple rare sugars for medicinal applications may be possible.

A Gram-negative, rod-shaped, motile and strictly aerobic bacterium, designated NBU1469T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1469T grew optimally at temperature of 40ºC, NaCl concentration of 2.0% (w/v) and pH 7.5. Catalase and oxidase activities, H2S production, nitrate reduction and hydrolysis of Tween 20 were positive. Indole, methyl red reaction, urease, hydrolysis of gelatin, starch, casein, Tweens 40, 60 and 80 were negative. The major cellular fatty acids were C16:0, C19:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The only respiratory quinone was ubiquinone-10 (Q-10). The major polar lipids were phosphatidylglycerol, two unidentified amino-phospholipids and two unidentified phospholipids. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Nisaea nitritireducens DR41_18T (98.1%) and Nisaea denitrificans DR41_21T (97.6%). Phylogenetic analyses indicated that strain NBU1469T formed a distinct lineage with strains Nisaea nitritireducens DR41_18T and Nisaea denitrificans DR41_21T within the genus Nisaea. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1469T and related species of genus Nisaea were well below the threshold limit for prokaryotic species delineation. The DNA G+C content was 63.6 mol%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1469T is considered to be a representative of a novel species in the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed. The type strain is NBU1469T (=KCTC 82224T=MCCC 1K04763T).

Hierarchical flower-like NiMnO3 composites were facilely prepared by template-free hydrothermal treatment and calcined under mild conditions. Due to the strong ability to absorb oxygen, the as-prep...

The genus Pseudonocardia belongs to a group of actinomycetes, and is a member of the family Pseudonocardiacea. The members of this genus are aerobic, Gram-positive, non-motile bacteria that are commonly found in soil, plant, and environment. Although this genus has a low clinical significance, however it has an important role in biotechnology due to the production of secondary metabolites, some of which have anti-bacterial, anti-fungal, and anti-tumor effects. The use of phenotypic tests such as gelatinase activity, starch hydrolysis, catalase, and oxidase tests, as well as molecular methods, such as polymerase chain reaction (PCR), are necessary for Pseudonocardia identification at the genus and species levels.

In this work, we report a simple electrochemical sensor that detects proteinuria upon immersion into a sample. We immobilized peptidases in conjunction with glutamate oxidase (GluOx) on a Prussian blue-modified glassy carbon (PB-GC) electrode. In the sensor, albumin, which is a major protein in urine, was degraded by peptidases to produce glutamate that was detected by GluOx on PB-GC. Initially, we validated the strategy for the detection of proteinuria using glutamate sensing. We quantified the amount of glutamate produced by degrading human serum albumin with HCl and carboxypeptidase A (CPA). The results indicated that an increase in the enzyme degradation rate was necessary for daily proteinuria sensing that needs to be completed within 1 h. Therefore, we investigated the use of endopeptidase in conjunction with CPA. The combination of proteinase K and CPA liberated glutamate from albumin at 17.5 times higher comparing to solely CPA. The subsequent glutamate assay using peptidase-modified GluOx-PB-GC electrode did not exhibit changes in the electrochemical signal with increases in the glutamate concentration, because peptidase degraded the GluOx on the modified electrode. To prevent this, GluOx was encapsulated in ZIF-8. Glutamate was successfully detected using peptidase and GluOx encapsulated in the ZIF-8-modified PB-GC (peptidase-ZIF-8/GluOx-PB-GC) electrode. Finally, an albumin assay was performed using the peptidase-ZIF-8/GluOx-PB-GC electrode, wherein albumin was detected with a sensitivity of 0.1 mg/mL within 30 min. Our simple and easy-to-use method for albumin detection provides a viable sensor for daily urinalysis.

Tomato fruit are sensitive to chilling injury (CI) during cold storage. Several factors have been discovered to be involved in chilling injury of tomato fruit. Plant hormones play an important regulatory role, however, the relationship between chilling injury and N6-methyladenosine (m6A) methylation of transcripts in plant hormone pathways has not been reported yet. In order to clarify the complex regulatory mechanism of m6A methylation on chilling injury in tomato fruit, Nanopore direct RNA sequencing was employed. A large number of enzymes and transcription factors were found to be involved in the regulation process of fruit chilling injury, which were associated with plant hormone, such as 1-aminocyclopropane 1-carboxylate synthase (ACS), aspartate aminotransferase (AST), auxin response factor (ARF2), ethylene response factor 2 (ERF2), gibberellin 20-oxidase-3 (GA20ox) and jasmonic acid (JA). By conjoint analysis of the differential expression transcripts related to chilling injury and m6A methylation differential expression transcripts 41 differential expression transcripts were identified involved in chilling injury including 1-aminocyclopropane-1-carboxylate oxidase (ACO) and pectinesterase (PE) were down-regulated and heat shock cognate 70 kD protein 2 (cpHSC70), HSP70-binding protein (HspBP) and salicylic acid-binding protein 2 (SABP2) were up-regulated. Our results will provide a deeper understanding for chilling injury regulatory mechanism and post-harvest cold storage of tomato fruit.

Background Developing new pesticides with multi-function may be a suitable strategy to save time and cost and reduce the emergence of resistant strains of pests and pathogens. The organophosphorus derivatives have not been widely used in agriculture as fungicides. In this work, a series of six α-amino phosphonate derivatives were prepared and tested for their fungicidal and anti-phenol oxidase activities. Results The prepared compounds revealed a promising anti-fungal activity against Macrophomina phaseolina and Pythium aphanidermatum, especially M4, with MIC of 62 mg/L for M. phaseolina. M4 did not affect the fungus permeability rate of the cell membrane; however, it displayed a significant efficiency on mycelial soluble protein content. M4 and M3 with a hydroxyl group on the aniline moiety exhibited an observed anti-phenol oxidase activity. M4 inhibited the enzyme at 1 mg/mL. The DFT theoretical study revealed a significant correlation between the substituents of aniline moiety and the bioactivity of the studied compounds. The negative charge conspicuously influenced the anti-phenol oxidase activity. Conclusions Our findings suggest the studied compounds as bases to design more effective α-amino phosphonate fungicides with additional anti-phenol oxidase activity. Graphic abstract

The crystal structure of l -lactate oxidase in complex with l -lactate was solved at a 1.33 A resolution. The electron density of the bound l -lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l -lactate was bound to the FMN and an additional active site within the enzyme complex. l -lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l -lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.

Engineering of enzyme microenvironment can surprisingly boost the apparent activity. However, the underlying regulation mechanism is not well-studied at a molecular level so far. Here, we present a modulation of two model enzymes of cytochrome c (Cty C) and d-amino acid oxidase (DAAO) with opposite pH-activity profiles using ionic polymers. The operational pH of poly (acrylic acid) modified Cyt C and polyallylamine modified DAAO was extended to 3–7 and 2–10 where the enzyme activity was larger than that at their optimum pH of 4.5 and 8.5 by 106% and 28%, respectively. The cascade reaction catalyzed by two modified enzymes reveals a 1.37-fold enhancement in catalytic efficiency compared with their native counterparts. The enzyme activity boosting is understood by performing the UV–vis/CD spectroscopy and molecular dynamics simulations in the atomistic level. The increased activity is ascribed to the favorable microenvironment in support of preserving enzyme native structures nearby cofactor under external perturbations.

Carbon dots (C-dots) are attractive for their low cost, excellent optical properties, and low toxicity, but most C-dots are limited by their UV absorbance, especially for photodynamic therapy (PDT) applications. Developing rational and simple strategies to obtain C-dots with longer excitation/emission wavelengths remains a challenge. Previous work showed that C-dots can produce a high yield of reactive oxygen species (ROS) upon UV light illumination. We herein show that the produced ROS can lead to further oxidation and growth of C-dots, leading to visible light absorption (400 nm–650 nm) and extend fluorescence emission from blue to green. In addition, the light-treated C-dots showed boosted peroxidase and oxidase mimicking activity than the initial C-dots in the absence of light. In the presence of visible light, the catalytic efficiency of the light-treated C-dots is 11-fold much higher than the pristine C-dots. The light-treated C-dots can be used as a PDT agent due to the extended excitation and visible photo-oxidative activity, simultaneously providing a highly efficient cancer therapy. This study provides a simple way to modify C-dots leading to novel properties.

Aerobic respiration is a key energy-producing pathway in many prokaryotes and virtually all eukaryotes. The final step of aerobic respiration is most commonly catalyzed by heme-copper oxidases embedded in the cytoplasmic or mitochondrial membrane. The majority of these terminal oxidases contain a prenylated heme (typically heme a or occasionally heme o) in the active site. In addition, many heme-copper oxidases, including mitochondrial cytochrome c oxidases, possess a second heme a cofactor. Despite the critical role of heme a in the electron transport chain, the details of the mechanism by which heme b, the prototypical cellular heme, is converted to heme o and then to heme a remain poorly understood. Recent structural investigations, however, have helped clarify some elements of heme a biosynthesis. In this review, we discuss the insight gained from these advances. In particular, we present a new structural model of heme o synthase (HOS) based on distance restraints from inferred coevolutionary relationships and refined by molecular dynamics simulations that are in good agreement with the experimentally determined structures of HOS homologs. We also analyze the two structures of heme a synthase (HAS) that have recently been solved by other groups. For both HOS and HAS, we discuss the proposed catalytic mechanisms and highlight how new insights into the heme-binding site locations shed light on previously obtained biochemical data. Finally, we explore the implications of the new structural data in the broader context of heme trafficking in the heme a biosynthetic pathway and heme-copper oxidase assembly.

Nanozymes were the novel research field to replace natural enzymes because of stability and low cost. However, the research on nanozymes was mainly focused on peroxidase, and there was little research about nanozymes with oxidase-like activity, especially mimic oxidase of small molecules related to human physiology. High levels of uric acid (UA) in the body can cause hyperuricemia and gout. And natural uricase cured this disease because it could oxidize UA. The oxidase-like activity of mixed valence state metal organic frameworks with cerium (MVSM) had been studied, but MVSM was found to have uricase-like activity in this article. The catalytic process of UA with MVSM was studied by a variety of analytical methods, which was similar to the natural uricase except for further oxidation of H2O2. The catalytic activity constants of MVSM were acquired by the Michaelis-Menten equation. MVSM had a better ability to catalyze UA in in vivo and in vitro experiments. An enzyme-free analysis–based mimic uricase for UA was established. All the experimental results proved that MVSM had a good prospect to replace the natural uricase.

The plant‐parasitic nematode Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), causes enormous economic loss every year. Currently, little is known about the pathogenic mechanisms of PWD. Several effectors have been identified in B. xylophilus, but their functions and host targets have yet to be elucidated. Here, we demonstrated that BxSCD1 suppresses cell death and inhibits B. xylophilus PAMP BxCDP1‐triggered immunity in Nicotiana benthamiana and Pinus thunbergii. BxSCD1 was transcriptionally upregulated in the early stage of B. xylophilus infection. In situ hybridization experiments showed that BxSCD1 was specifically expressed in the dorsal glands and intestine. Cysteine residues are essential for the function of BxSCD1. Transient expression of BxSCD1 in N. benthamiana revealed that it was primarily targeted to the cytoplasm and nucleus. The morbidity was significantly reduced in P. thunbergii infected with B. xylophilus when BxSCD1 was silenced. We identified 1‐aminocyclopropane‐1‐carboxylate oxidase 1, the actual ethylene‐forming enzyme, as a host target of BxSCD1 by yeast two‐hybrid and coimmunoprecipitation. Overall, this study illustrated that BxSCD1 played a critical role in the B. xylophilus–plant interaction., BxSCD1 suppressed plant immunity in Nicotiana benthamiana and Pinus thunbergii. We report for the first time that BxSCD1 interacts with an ethylene‐forming enzyme in pine.

Whole-cell systems offer many benefits for biochemical production, such as relatively easy enzyme control and higher tolerance toward harsh environments, than purified enzymes. These systems can be applied to many bioconversion reactions, but they sometimes require cofactor regeneration units to support reactions at high substrate concentrations. Here, we examined l-glutamate oxidase (GOX) from Streptomyces sp. X119-6, which produces α-ketoglutarate (α-KG) from l-glutamate, and catalase (KatE) from Escherichia coli, which removes hydrogen peroxide generated by GOX. After optimizing the expression vector, pH, strains, culture conditions, and isopropyl β-d-1-thiogalactopyranoside concentration, we compared their efficiency to that of a previously reported GOX from Streptomyces mobaraensis. Our results indicated that GOX from Streptomyces sp. X119-6 and KatE increased α-KG production by 2.76-fold. This GOX required high levels of α-KG as an amino donor to convert 5-aminovaleric acid to glutaric acid. Performing the reaction at pH 8 enabled us to avoid the exogenous addition of catalase, but severe substrate inhibition was observed, resulting in the production of 287 mM glutaric acid. This α-KG regeneration system has potential for improving production in various aminotransferase systems.

A Gram-stain-negative, rod-shaped (0.3–0.6 × 0.9–2.2 µm), facultatively aerobic, non-motile and yellow-coloured bacterium, designated strain F6397T, was isolated from a marine sediment in Weihai, PR China. Growth of strain F6397T occurred at 4–37 °C (optimum, 30–33 °C), pH 6.0–9.0 (optimum, 7.5) and in the presence of 1–12% (optimum, 3.0%) (w/v) NaCl. Strain F6397T showed oxidase- and catalase-positive activities. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the strain was assigned to the genus Winogradskyella. Strain F6397T exhibited 95.5–98.1% sequence similarities to recognized species of the genus Winogradskyella. The average nucleotide identity (ANI) scores with Winogradskyella ludwigii HL116T, Winogradskyella litoriviva KMM 6491 T and Winogradskyella thalassocola KMM 3907 T were 80.1, 78.9 and 82.6%, respectively. The digital DNA–DNA hybridization (dDDH) scores were 23.5, 22.9 and 25.7%, respectively. The genomic DNA G + C content was 33.5 mol%. Strain F6397T contained iso-C15:0, iso-C15:1G, iso-C15:0 3-OH and iso-C17:0 3-OH as the predominant fatty acid and MK-6 as the predominant menaquinone. The polar lipid profiles contained phosphatidylethanolamine, three unidentified aminolipids and four unidentified lipids. On the basis of the evidence presented in this study, strain F6397T is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella marina sp. nov. is proposed. The type strain is F6397T (= KCTC 82422 T = MCCC 1H00438T).

AIMS The persipeptides were recognized as a promising source of multiple pharmaceutical activities which were revealed following structure-activity prediction and examination in experimental analysis. METHODS AND RESULTS The profile of toxicity, antioxidant, anti-inflammatory, anti-diabetic and anti-ageing activity of persipeptides and the crude extract were evaluated experimentally. The pure Persipeptide A and B revealed a moderate xanthine oxidase inhibition activity at the concentration of 10 µg/ml. Persipeptide exhibited α-glucosidase inhibition activity (~10% inhibition) and less than 2% tyrosinase inhibition activity at the concentration of 10 µg/ml. The extract exhibited the inhibition of less than 2% acetylcholine esterase inhibition activity, but the pure persipeptide showed 6%-14% inhibition activity at the concentration of 10 µg/ml. The molecular docking analysis revealed that the activities of Persipeptide A and B are due to interaction with xanthin oxidase, α-amylase, α-glucosidase, tyrosinase and acetylcholine esterase enzymes. CONCLUSIONS The persipeptides showed a similar inhibition rate with positive control that might imply its potential as an anti-diabetic and anti-gout compound among. Only acetylcholine esterase inhibition of persipeptide was higher than the extract. The interacting amino acids of the molecules with different targets show that persipeptides might have antioxidant, anti-inflammatory, anti-diabetic, anti-ageing activity and even other potential pharmaceutical activities that were not investigated in this study. SIGNIFICANCE AND IMPACT OF THE STUDY This report was presented to find some new pharmaceutical activities of Persipeptide A and B including the α-glucosidase inhibition activity as a molecular target of diabetes mellitus. Persipeptides also exhibited an effective inhibition of xanthine oxidase (XO) which can be a drug-like candidate in the treatment of diseases associated with XO like gout. The binding values indicated the interaction of persipeptides with these enzymes.

Anaerobic ammonium oxidation (anammox) is a bacterial process in which ammonium and nitrite are combined into dinitrogen gas and water, yielding energy for the cell. This process relies on a series of redox reactions catalyzed by a set of enzymes, with electrons being shuttled to and from these enzymes, likely by small cytochrome c proteins. For this system to work productively, these electron carriers require a degree of specificity toward the various possible redox partners they encounter in the cell. Here, we compare two cytochrome c proteins from the anammox model organism Kuenenia stuttgartiensis. We show that they are highly homologous, are expressed at comparable levels, share the same fold, and display highly similar redox potentials, yet one of them accepts electrons from the metabolic enzyme hydroxylamine oxidase (HAO) efficiently, whereas the other does not. An analysis of the crystal structures supplemented by Monte Carlo simulations of the transient redox interactions suggests that this difference is at least partly due to the electrostatic field surrounding the proteins, illustrating one way in which the electron carriers in anammox could attain the required specificity. Moreover, the simulations suggest a different “outlet” for electrons on HAO than has traditionally been assumed.

Holothurians referred to as “teatfish” contain three species, Holothuria (Microthele) nobilis, H. (M.) whitmaei and H. (M.) fuscogilva, that have recently been included in CITES Appendix II for sustainable catch and trade. Taxonomic key traits of these species are primarily genetic sequences and there are few morphological differences among them, except for color patterns of the body. Although located in the northern limit of their distribution, there have been no reports identifying species of teatfish in Japan based on both morphology and genetic traits. This study investigated sequences of mitochondrial cytochrome oxidase subunit I (COI) and morphology including ossicles of black and white teatfish collected from Okinawa Island and Yaeyama Islands, Japan. The entirely black specimens and white specimens having black mottles on the dorsal side were identified as Holothuria (M.) whitmaei and H. (M.) fuscogilva, respectively, based on the agreement of genetic and morphological traits and with published data. There is no clear difference in COI sequences between specimens from Japan and other Pacific regions for both species. The only inconsistent finding with previous reports was that one specimen of H. (M.) whitmaei lacked Cuvierian tubules, which are usually present in this species, implying plasticity of this trait. Mean diameter of the disc and height of the pillars of table ossicles were shorter and longer, respectively, in H. (M.) fuscogilva than in H. (M.) whitmaei (p

The monocot lineage-specific miR528 was previously established as a multistress regulator. However, it remains largely unclear how miR528 participates in response to salinity stress in rice. Here, we show that miR528 positively regulates rice salt tolerance by down-regulating a gene encoding l-ascorbate oxidase (AO), thereby bolstering up the AO-mediated abscisic acid (ABA) synthesis and ROS scavenging. Overexpression of miR528 caused a substantial increase in ascorbic acid (AsA) and ABA contents but a significant reduction in ROS accumulation, resulting in the enhanced salt tolerance of rice plants. Conversely, knockdown of miR528 or overexpression of AO stimulated the expression of the AO gene, hence lowering the level of AsA, a critical antioxidant that promotes the ABA content but reduces the ROS level, and then compromising rice tolerance to salinity. Together, the findings reveal a novel mechanism of the miR528-AO module-mediated salt tolerance by modulating the processes of AsA and ABA metabolism as well as ROS detoxification, which adds a new regulatory role to the miR528-AO stress defense pathway in rice.

Flavin as a cofactor is an extremely versatile molecule that participates in a wide range of biochemical reactions. A special characteristic of the flavin cofactor, unique for a metal free cofactor, is its ability to react in its reduced form with molecular oxygen. This is exploited in flavoprotein oxidases and monooxygenases. The flavin-mediated reduction of dioxygen can lead to various reactive oxygen species (ROS), primarily hydrogen peroxide and superoxide. No systematic analysis of the formation of such reduced oxygen species produced by flavoprotein oxidases and monooxygenases had been performed before. In this work, we investigated which ROS are formed by several prototype flavoprotein oxidases and monooxygenases: Phenylacetone monooxygenase (TfPAMO), an engineered TfPAMO variant that acts as an NADPH oxidase (TfPAMO C65D), eugenol oxidase (RjEUGO), and 5-hydroxymethyl furfural oxidase (MHMFO). The formed amounts of superoxide and hydrogen peroxide were measured under various conditions (different substrate concentrations, pH values and cosolvents). Interestingly, all flavoenzymes were found to produce, except for hydrogen peroxide, significant amounts of superoxide. Moreover, increased superoxide levels were measured at higher pH, which could be indicative for a pH-sensitive caged radical pair dissociation. To probe the effect of ROS formation on biocatalytic performance, conversions catalyzed by TfPAMO or RjEUGO, with or without catalase, were monitored. This revealed that catalase has a beneficial effect. No detrimental effect of the accumulation of superoxide on biocatalysis could be demonstrated. The results reveal that formation of ROS by flavoenzymes is highly dependent on the experimental conditions used. The results provide a better insight into the mechanism by which ROS is formed in flavoenzymes and may help studies or applications in which ROS formation should be promoted or minimized for industrial biocatalysis.

A Marinomonas-like, Gram-stain-negative, strictly aerobic and rod to ovoid-shaped bacterium, designated as strain A79T, was isolated from the seawater mixtures of oyster shells and brown algae in a coastal intertidal zone of Zhoushan, China. The strain was positive for oxidase and catalase. Colonies grown on marine agar for 48 h were round, milky white, smooth and moist with the diameter of 2–3 mm. Growth was observed at 15–30 °C (optimum, 25℃), pH 5.5–9.5 (optimum, pH 8.5) and with 0.5–8% (w/v) NaCl (optimum, 2–2.5%). The G + C content based on the genome sequence was 46.0%. The only respiratory quinone was Q-8. The main polar lipids contained phosphatidylglycerol, phosphatidylethanolamine, unidentified glycolipids, unidentified phospholipid and three unidentified lipids. The major fatty acids (> 10%) were C16:0, Summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c). The 16S rRNA gene sequence similarity between strain A79T and Marinomonas pollencensis IVIA-Po-185T was 97.4%, the similarities with other type strains of the genus Marinomonas were 93.8–96.7%. Based on the results, Marinomonas vulgaris sp. nov. was proposed as a novel species. The type strain is A79T (= MCCC 1K05799T = KCTC 82519T = JCM 34473T).

During food digestion, the intestinal epithelial barrier is essential for health. The intestinal epithelial is typically maintained by normal tight junctions. Barrier dysfunction and increased permeability have been observed for many diseases and, more recently, after ingestion of nitrogen contaminants such as nitrogen heterocycles, amine, and nitro. Mechanisms by which these contaminants disrupt the intestinal epithelial barrier remain poorly understood. Here, we review the chemical disruption of intestinal epithelium permeability of tight junction proteins, to propose a common mechanism involving the production of reactive oxygen species (ROS) followed by inflammation. Specifically, nitrogen-containing chemicals promote ROS generation, which disrupts the activity of the metabolic enzyme cytochrome P450s, leading to the activation of nicotinamide adenine dinucleotide phosphate oxidase.

Replacement of enzymes with nanomaterials such as atomically dispersed metal catalysts is one of the most crucial steps in addressing the challenges in biocatalysis. Despite the breakthroughs of single-atom catalysts in enzyme-mimicking, a fundamental investigation on the development of an instructional strategy is still required for mimicking biatomic/multiatomic active sites in natural enzymes and constructing synergistically enhanced metal atom active sites. Herein, Fe2NC catalysts with atomically dispersed Fe-Fe dual-sites supported by the metal-organic frameworks-derived nitrogen-doped carbon are employed as biomimetic catalysts to perform proof-of-concept investigation. The effect of Fe atom number toward typical oxidase (cytochrome C oxidase, NADH oxidase, and ascorbic acid oxidase) and peroxidase (NADH peroxidase and ascorbic acid peroxidase) activities is systematically evaluated by experimental and theoretical investigations. A peroxo-like O2 adsorption in Fe2NC nanozymes could accelerate the O-O activation and thus achieve the enhanced enzyme-like activities. This work achieves the vivid simulation of the enzyme active sites and provides the theoretical basis for the design of high-performance nanozymes. As a concept application, a colorimetric biosensor for the detection of S2− in tap water is established based on the inhibition of enzyme-like activity of Fe2NC nanozymes.

Benefiting from the merits of high stability and superior activity, nanozymes are recognized as promising alternatives to natural enzymes. Despite the great leaps in the field of therapy and colorimetric sensing, the development of highly sensitive nanozyme-involved photoelectrochemical (PEC) biosensors is still in its infancy. Specifically, the investigation of multifunctional nanozymes facilitating different catalytic reactions remains largely unexplored due to the difficulty in synergistically amplifying the PEC signals. In this work, mesoporous trimetallic AuPtPd nanospheres were synthesized with both efficient oxidase and peroxidase-like activities, which can synergistically catalyze the oxidation of 4-chloro-1-naphthol to produce benzo-4-chlorohexadienone precipitation on the surface of photoactive materials, and thus lead to the decreased photocurrent as well as increased charge-transfer resistance. Inspired by the proton-dependent catalytic activity of nanozymes, a self-regulated dual-modal PEC and electrochemical bioassay of urease activity was innovatively established by in situ regulating the activity of AuPtPd nanozymes through urease-mediated proton-consuming enzymatic reactions, which can remarkably improve the accuracy of the assay. Meanwhile, the determination of urease activity in spiked human saliva samples was successfully realized, indicating the reliability of the biosensor and its application prospects in clinical diagnosis.