Flavin Monooxygenase Research Articles

Roles of selected non-P450 human oxidoreductase enzymes in protective and toxic effects of chemicals: review and compilation of reactions.

This is an overview of the metabolic reactions of drugs, natural products, physiological compounds, and other (general) chemicals catalyzed by flavin monooxygenase (FMO), monoamine oxidase (MAO), NAD(P)H quinone oxidoreductase (NQO), and molybdenum hydroxylase enzymes (aldehyde oxidase (AOX) and xanthine oxidoreductase (XOR)), including roles as substrates, inducers, and inhibitors of the enzymes. The metabolism and bioactivation of selected examples of each group (i.e., drugs, “general chemicals,” natural products, and physiological compounds) are discussed. We identified a higher fraction of bioactivation reactions for FMO enzymes compared to other enzymes, predominately involving drugs and general chemicals. With MAO enzymes, physiological compounds predominate as substrates, and some products lead to unwanted side effects or illness. AOX and XOR enzymes are molybdenum hydroxylases that catalyze the oxidation of various heteroaromatic rings and aldehydes and the reduction of a number of different functional groups. While neither of these two enzymes contributes substantially to the metabolism of currently marketed drugs, AOX has become a frequently encountered route of metabolism among drug discovery programs in the past 10–15 years. XOR has even less of a role in the metabolism of clinical drugs and preclinical drug candidates than AOX, likely due to narrower substrate specificity.

Deficiency of PSRC1 accelerates atherosclerosis by increasing TMAO production via manipulating gut microbiota and flavin monooxygenase 3

ABSTRACT Maladaptive inflammatory and immune responses are responsible for intestinal barrier integrity and function dysregulation. Proline/serine-rich coiled-coil protein 1 (PSRC1) critically contributes to the immune system, but direct data on the gut microbiota and the microbial metabolite trimethylamine N-oxide (TMAO) are lacking. Here, we investigated the impact of PSRC1 deletion on TMAO generation and atherosclerosis. We first found that PSRC1 deletion in apoE−/− mice accelerated atherosclerotic plaque formation, and then the gut microbiota and metabolites were detected using metagenomics and untargeted metabolomics. Our results showed that PSRC1 deficiency enriched trimethylamine (TMA)-producing bacteria and functional potential for TMA synthesis and accordingly enhanced plasma betaine and TMAO production. Furthermore, PSRC1 deficiency resulted in a proinflammatory colonic phenotype that was significantly associated with the dysregulated bacteria. Unexpectedly, hepatic RNA-seq indicated upregulated flavin monooxygenase 3 (FMO3) expression following PSRC1 knockout. Mechanistically, PSRC1 overexpression inhibited FMO3 expression in vitro, while an ERα inhibitor rescued the downregulation. Consistently, PSRC1-knockout mice exhibited higher plasma TMAO levels with a choline-supplemented diet, which was gut microbiota dependent, as evidenced by antibiotic treatment. To investigate the role of dysbiosis induced by PSRC1 deletion in atherogenesis, apoE−/− mice were transplanted with the fecal microbiota from either apoE−/− or PSRC1−/−apoE−/− donor mice. Mice that received PSRC1-knockout mouse feces showed an elevation in TMAO levels, as well as plaque lipid deposition and macrophage accumulation, which were accompanied by increased plasma lipid levels and impaired hepatic cholesterol transport. Overall, we identified PSRC1 as an atherosclerosis-protective factor, at least in part, attributable to its regulation of TMAO generation via a multistep pathway. Thus, PSRC1 holds great potential for manipulating the gut microbiome and alleviating atherosclerosis.

Complexity of the auxin biosynthetic network in Arabidopsis hypocotyls is revealed by multiple stable-labeled precursors

Auxin is a key regulator of plant development and in Arabidopsis thaliana can be synthesized through multiple pathways; however, the contributions of various biosynthetic pathways to specific developmental processes are largely unknown. To trace the involvement of various biosynthetic routes to indole-3-acetic acid (IAA) under conditions that induce adventitious root formation in Arabidopsis hypocotyls, we treated seedlings with three different stable isotope-labeled precursors ([13C6]anthranilate, [15N1]indole, and [13C3]serine) and monitored label incorporation into a number of proposed biosynthesis intermediates as well as IAA. We also employed inhibitors targeting tryptophan aminotransferases and flavin monooxygenases of the IPyA pathway, and treatment with these inhibitors differentially altered the labeling patterns from all three precursors into intermediate compounds and IAA. [13C3]Serine was used to trace utilization of tryptophan (Trp) and downstream intermediates by monitoring 13C incorporation into Trp, indole-3-pyruvic acid (IPyA), and IAA; most 13C incorporation into IAA was eliminated with inhibitor treatments, suggesting Trp-dependent IAA biosynthesis through the IPyA pathway is a dominant contributor to the auxin pool in de-etiolating hypocotyls that can be effectively blocked using chemical inhibitors. Labeling treatment with both [13C6]anthranilate and [15N1]indole simultaneously resulted in higher label incorporation into IAA through [15N1]indole than through [13C6]anthranilate; however, this trend was reversed in the proposed precursors that were monitored, with the majority of isotope label originating from [13C6]anthranilate. An even greater proportion of IAA became [15N1]-labeled compared to [13C6]-labeled in seedlings treated with IPyA pathway inhibitors, suggesting that, when the IPyA pathway is blocked, IAA biosynthesis from labeled indole may also come from an origin independent of the measured pool of Trp in these tissues.

Investigating the Mechanistic Strategies of the Two‐component FMN‐dependent Alkanesulfonate Monooxygenase Systems

The two‐component alkanesulfonate monooxygenase systems, consisting of a flavin reductase (SsuE and MsuE) and an alkanesulfonate monooxygenase (SsuD and MsuD), enable bacterial organisms to utilize a broad range of alkanesulfonates when sulfur is limiting. The flavin reductases supply reduced flavin to their partner monooxygenase for the desulfonation of sulfonated compounds through the formation of a flavin oxygenating intermediate. Commonly, flavin monooxygenases have been proposed to utilize a C4a‐(hydro)peroxyflavin as the oxygenating intermediate. However, unlike other flavin monooxygenase enzymes, this intermediate has not been spectrally observed in the alkanesulfonate monooxygenase enzymes. Recent studies reported the formation of a flavin‐N5‐oxide which forms as an intermediate during turnover or as the final product in some flavin monooxygenases. It is hypothesized that the alkanesulfonate monooxygenases could employ a flavin‐N5‐adduct based on amino acid sequence alignments and structural similarities with enzymes employing similar intermediates.SsuD and MsuD share comparable structural properties but have different specificities for alkanesulfonate substrates. Although the substrate preference is critical for catalytic competence, SsuD and MsuD likely utilize similar catalytic steps for desulfonation. Previous studies identified various polar and nonpolar amino acid residues that were critical for the formation and stabilization of the flavin‐N5‐oxide. The conserved nonpolar amino acids are proposed to control the interaction of the flavin‐N5 with molecular oxygen, whereas the polar amino acids are involved in stabilizing the superoxide anion involved in the formation of the flavin‐N5 oxygenating intermediate. Some of these amino acids were also found to be conserved in the alkanesulfonate monooxygenases and their role was further evaluated through site‐directed mutagenesis. The V108T and T109A SsuD variants had similar activity to wild‐type SsuD; however, the N108L SsuD variant had no measurable activity. These results support the role of Asn in stabilizing the proposed flavin‐N5 oxygenating intermediate. Since a flavin‐N5‐oxide has been identified as an intermediate or as the final product in some two‐component monooxygenases, HPLC and mass spectrometric analyses were performed to determine if the alkanesulfonate monooxygenases form the flavin‐N5‐oxide. To provide evidence for kinetic steps and identify flavin‐N5 reaction intermediates, stopped‐flow kinetic experiments were performed with wild‐type and variants of SsuD and MsuD. The findings from this study provide insight into the mechanistic features of the alkanesulfonate monooxygenases as well as their role in the overall global sulfur cycle.

Elucidating The Nicotinic Acid Degradation Pathway In Bacillus niacini: Identification and Biochemical Characterization Of Proteins Of Unknown Function

N‐Heterocyclic aromatic compounds (NHACs) are abundant in the manufacturing of solvents, pesticides, and dyes; however, accumulation of these compounds can lead to detrimental environmental impacts. To alleviate these impacts, it is critical to understand the mechanisms microbial species use to catabolize these types of molecules so that bioremediation techniques can be implemented. Nicotinic acid is commonly used as a model system for NHAC degradation because it is environmentally safe and can be degraded by a variety of bacterial species. Bacillus niacini is a soil‐dwelling bacterium that has been identified to degrade NA through an uncharacterized pathway. This makes B. niacini an organism of interest, as elucidation of the mechanism by which it catabolizes NA will reveal differences between these pathways that can be exploited to optimize bioremediation efforts. Bioinformatic analyses of genomic and transcriptomic data revealed a potential operon responsible for NA degradation in B. niacini. This operon contains 15 genes that encode for 7 enzymes that are necessary for NA degradation as well as three genes that have not yet been elucidated: a domain of unknown function (DUF), a flavin monooxygenase (FMO), and a hypothetical protein (HP). NA degradation in B. niacini is unique in that it involves two ring hydroxylating steps, forming the intermediate 2,6‐dihydroxynicotinic acid (DHNA). After decarboxylation of DHNA to 2,6‐dihydroxypridine (DHP), we show that the DUF hydroxylates DHP to 2,3,6‐trihydroxypyridine (THP). Separation of the DUF‐catalyzed reaction products by C‐18 chromatography (HPLC) and detection by UV‐Vis at pH 3 shows a new species with an Absmaxat 320 nm and 590 nm. Mass spectrometry (Q‐TOF) analysis of the reaction identifies the products as DHP‐ and THP‐ oxidized dimers (nicotine blue). Formation of these products are observed to be O2 dependent. When the DUF, FMO and HP genes are recombinantly expressed together in E. coli, the cell extract is able to degrade DHP without forming the DHP‐oxidized dimer. That observation suggests the possible roles of FMO and/or HP in catalyzing the conversion of the DHP‐dimer back to DHP. This study is the first to characterize the genes responsible for NA degradation by B. niacini and further establishes the NA degradation pathway.

Flavin binding affinity and initial kinetic characterization of DnmZ, a flavin‐dependent N‐oxygenase

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius. It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a derivatized form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes a sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases.Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 mM, which compares well with other Class D monooxygenases. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination but has no effect on the intermediate’s formation. We are also investigating the effect of mutating active site residues on C4a‐OOH formation and elimination.

Plant biology: Local auxin synthesis drives pollen maturation in barley

Pollen grains stock up on starch to power germination and pollen tube growth upon pollination. New findings in barley show that localized auxin biosynthesis by a YUC flavin monooxygenase leads to reprogramming energy metabolism during pollen maturation.

The PH/MyTH4/FERM molecule MAX-1 inhibits UNC-5 activity in the regulation of VD growth cone protrusion in Caenorhabditis elegans.

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of Caenorhabditis elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP, and UNC-34/Enabled. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis.

Transcriptome Analysis Reveals Genetic Factors Related to Callus Induction in Barley

Barley is an important cereal crop worldwide. Its genetic transformation is now limited to very few cultivars because of the high genotype dependence of embryogenic callus. To reveal the key genes or factors controlling the callus induction and plantlet regeneration in barley, we compared the transcriptomic profiles of immature embryos of Golden Promise and ZU9, which differed dramatically in the efficiency of the genetic transformation. The samples were taken at 0, 5, 10 and 20 days of the culture, respectively. In total, 5386 up-regulated and 6257 down-regulated genes were identified in Golden Promise. Several genes, identified exclusively in GP callus, were selected for further investigation. These genes were mainly involved in protein metabolism, energy metabolism, stress response, detoxification and ubiquitin–proteasome. Four YUCCA flavin monooxygenases, one PIN-FORMED, one tryptophan aminotransferase related, three small auxin up RNA, three indole-3-acetic acid and one adenylate isopentenyl transferase, seven cytokinin oxidase/dehydrogenase, three Arabidopsis histidine kinase, three Arabidopsis histidine phosphotransfer protein, and one Arabidopsis response regulator were differentially expressed in the calli of the two barley genotypes, suggesting that biosynthesis, response and transport of auxin and cytokinin might be associated with cell reprogramming during callus induction. The current results provide insights into molecular mechanisms of callus induction at an early developmental stage and are helpful for optimizing the tissue culture system in barley.

Bifidobacterium breve and Bifidobacterium longum Attenuate Choline-Induced Plasma Trimethylamine N-Oxide Production by Modulating Gut Microbiota in Mice.

Atherosclerosis is the main cause of myocardial infarction and stroke, and the morbidity and mortality rates of cardiovascular disease are among the highest of any disease worldwide. Excessive plasma trimethylamine-N-oxide (TMAO), an intestinal metabolite, promotes the development of atherosclerosis. Therefore, effective measures for reducing plasma TMAO production can contribute to preventing atherosclerosis. Probiotics are living microorganisms that are beneficial to the human body, and some of them can attenuate plasma TMAO production. To explore the effects of probiotic supplementation on plasma TMAO in choline-fed mice, we intragastrically administered eight strains of Bifidobacterium breve and eight strains of Bifidobacterium longum to mice for 6 weeks. B. breve Bb4 and B. longum BL1 and BL7 significantly reduced plasma TMAO and plasma and cecal trimethylamine concentrations. However, hepatic flavin monooxygenase (FMO) activity, flavin-containing monooxygenase 3 (FMO3), farnesoid X receptor (FXR) protein expression and TMAO fractional excretion were not significantly affected by Bifidobacterium supplementation. The treatment of Bifidobacterium strains modulated the abundances of several genera such as Ruminococcaceae UCG-009, Ruminococcaceae UCG-010, which belong to the Firmicutes that has been reported with cut gene clusters, which may be related to the reduction in intestinal TMA and plasma TMAO. Additionally, a reduction in Ruminococcaceae indicates a reduction in circulating glucose and lipids, which may be another pathway by which Bifidobacterium strains reduce the risk of atherosclerosis. The effect of Bifidobacterium strains on Bacteroides also suggests a relationship between the abundance of this genus and TMA concentrations in the gut. Therefore, the mechanism underlying these changes might be gut microbiota regulation. These Bifidobacterium strains may have therapeutic potential for alleviating TMAO-related diseases.

Experimental Periodontitis Deteriorated Atherosclerosis Associated With Trimethylamine N-Oxide Metabolism in Mice.

BackgroundPeriodontitis is considered a risk factor for atherosclerosis, but the mechanism is not clear. It was reported that oral administration of Porphyromonas gingivalis altered the gut microbiota in mice. Gut dysbiosis and the intestinal metabolite trimethylamine N-oxide (TMAO) were verified to be associated with atherosclerosis. Therefore, the possible TMAO-related mechanism between periodontitis and atherosclerosis needs to be explored.MethodsExperimental periodontitis was established by oral administration of P. gingivalis for 2 months in ApoE−/− mice. Mouse hemi-mandibles were scanned using Micro-CT. Quantification of TMAO was performed using liquid chromatography–tandem mass spectrometry. Mouse feces were collected and the bacterial DNA was extracted, then the gut microbiota was analyzed using 16S rRNA genes. Atherosclerotic lesion areas were quantified. Livers, small intestines, and large intestines were analyzed for gene expression.ResultsAggravated atherosclerosis plaques were found in experimental periodontitis mice. Plasma TMAO, a pathogenic factor of atherosclerosis, was initially found to be increased in periodontitis mice. Changes in the composition and abundance of the intestinal microflora of periodontitis mice were found. Flavin monooxygenase 3 (FMO3), the catalyzing enzyme of TMAO in the liver, was significantly increased, accompanied by an increase of IL-6 in liver, the abnormal intestinal integrity and enhanced plasma LPS. The IL-6 and LPS were verified to be able to increase FMO3 in HepG2 cells.ConclusionOur research discovered that experimental periodontitis in ApoE−/− mice induced gut dysbiosis and an increase in TMAO. These results suggest a possible mechanism by which periodontitis may accelerate atherosclerosis by influencing the intestinal microbes and the metabolism, which were triggered by inflammation of the liver and intestine.

A Review of The Contribution of Gut-Dependent Microbiota Derived Marker, Trimethylamine N-oxide (TMAO), in Coronary Artery Disease

Coronary artery disease (CAD) has a high prevalence and one of the principal drivers of mortality worldwide. Therefore, there is a requirement to develop sensitive diagnostic biomarkers, disease progression control and therapeutic stratification in order to keep a check on the disease rate. Atherosclerosis is a systemic disease, the main cause of heart disease, is associated with hyperlipidemia and lipid oxidation and has always been a common single leading cause of death in well-developed countries. In the attempts to study CAD and the causative agents for the disease, a metabolite circulating in the plasma termed trimethylamine-N-oxide (TMAO) has been found out to be an independent risk factor that increases CAD risk. The use of a metabolomic approach has proven useful in the recent past, as it can aid in the identification and quantification of several metabolites that play a crucial role for diagnosis and exploring therapeutic targets. TMAO is majorly synthesized by a process which involves the bioconversion of gut microbiota and hepatic flavin monooxygenases (FMOs) from nutrient-containing dietary trimethylamine (TMA). TMA is synthesized by gut bacterial fermentation from the components present in meat such as phosphatidylcholine (PC), betaine, choline, and L-carnitine. It can accentuate the process of atherosclerosis through the novel meta-organismal metabolic pathway. TMAO leads to atherogenesis by increasing vascular inflammation, reducing vascular functions and disrupting cholesterol homeostasis at various levels. This review article attempts to summarize the pool of evidence collected on the microbiota-dependent TMAO and its association with atherosclerosis. We performed literature search with Medline, PubMed, and Google Scholar, on “TMAO in CAD”, “metabolites in CAD” and “TMAO in other diseases” from the year 1990 to 2020. Although the circulatory TMAO has been identified as an independent marker for CAD, there is still no conclusive evidence to justify its role as a routine marker for CAD diagnosis. Future research must clarify the mechanisms which underpin these complex associations to determine if there is a causal link exists between TMAO and CAD.

Metopimazine is primarily metabolized by a liver amidase in humans.

Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first‐pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin‐monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP.

H2O2 Functions as a Downstream Signal of IAA to Mediate H2S-Induced Chilling Tolerance in Cucumber.

Hydrogen sulfide (H2S) plays a crucial role in regulating chilling tolerance. However, the role of hydrogen peroxide (H2O2) and auxin in H2S-induced signal transduction in the chilling stress response of plants was unclear. In this study, 1.0 mM exogenous H2O2 and 75 μM indole-3-acetic acid (IAA) significantly improved the chilling tolerance of cucumber seedlings, as demonstrated by the mild plant chilling injury symptoms, lower chilling injury index (CI), electrolyte leakage (EL), and malondialdehyde content (MDA) as well as higher levels of photosynthesis and cold-responsive genes under chilling stress. IAA-induced chilling tolerance was weakened by N, N′-dimethylthiourea (DMTU, a scavenger of H2O2), but the polar transport inhibitor of IAA (1-naphthylphthalamic acid, NPA) did not affect H2O2-induced mitigation of chilling stress. IAA significantly enhanced endogenous H2O2 synthesis, but H2O2 had minimal effects on endogenous IAA content in cucumber seedlings. In addition, the H2O2 scavenger DMTU, inhibitor of H2O2 synthesis (diphenyleneiodonium chloride, DPI), and IAA polar transport inhibitor NPA reduced H2S-induced chilling tolerance. Sodium hydrosulfide (NaHS) increased H2O2 and IAA levels, flavin monooxygenase (FMO) activity, and respiratory burst oxidase homolog (RBOH1) and FMO-like protein (YUCCA2) mRNA levels in cucumber seedlings. DMTU, DPI, and NPA diminished NaHS-induced H2O2 production, but DMTU and DPI did not affect IAA levels induced by NaHS during chilling stress. Taken together, the present data indicate that H2O2 as a downstream signal of IAA mediates H2S-induced chilling tolerance in cucumber seedlings.

Mixed Transcriptome Analysis Revealed the Possible Interaction Mechanisms between Zizania latifolia and Ustilago esculenta Inducing Jiaobai Stem-Gall Formation.

The smut fungus Ustilago esculenta infects Zizania latifolia and induces stem expansion to form a unique vegetable named Jiaobai. Although previous studies have demonstrated that hormonal control is essential for triggering stem swelling, the role of hormones synthesized by Z. latifolia and U. esculenta and the underlying molecular mechanism are not yet clear. To study the mechanism that triggers swollen stem formation, we analyzed the gene expression pattern of both interacting organisms during the initial trigger of culm gall formation, at which time the infective hyphae also propagated extensively and penetrated host stem cells. Transcriptional analysis indicated that abundant genes involving fungal pathogenicity and plant resistance were reprogrammed to maintain the subtle balance between the parasite and host. In addition, the expression of genes involved in auxin biosynthesis of U. esculenta obviously decreased during stem swelling, while a large number of genes related to the synthesis, metabolism and signal transduction of hormones of the host plant were stimulated and showed specific expression patterns, particularly, the expression of ZlYUCCA9 (a flavin monooxygenase, the key enzyme in indole-3-acetic acid (IAA) biosynthesis pathway) increased significantly. Simultaneously, the content of IAA increased significantly, while the contents of cytokinin and gibberellin showed the opposite trend. We speculated that auxin produced by the host plant, rather than the fungus, triggers stem swelling. Furthermore, from the differently expressed genes, two candidate Cys2-His2 (C2H2) zinc finger proteins, GME3058_g and GME5963_g, were identified from U. esculenta, which may conduct fungus growth and infection at the initial stage of stem-gall formation.

Environmental Occurrence, Toxicity Concerns, and Degradation of Diazinon Using a Microbial System.

Diazinon is an organophosphorus pesticide widely used to control cabbage insects, cotton aphids and underground pests. The continuous application of diazinon in agricultural activities has caused both ecological risk and biological hazards in the environment. Diazinon can be degraded via physical and chemical methods such as photocatalysis, adsorption and advanced oxidation. The microbial degradation of diazinon is found to be more effective than physicochemical methods for its complete clean-up from contaminated soil and water environments. The microbial strains belonging to Ochrobactrum sp., Stenotrophomonas sp., Lactobacillus brevis, Serratia marcescens, Aspergillus niger, Rhodotorula glutinis, and Rhodotorula rubra were found to be very promising for the ecofriendly removal of diazinon. The degradation pathways of diazinon and the fate of several metabolites were investigated. In addition, a variety of diazinon-degrading enzymes, such as hydrolase, acid phosphatase, laccase, cytochrome P450, and flavin monooxygenase were also discovered to play a crucial role in the biodegradation of diazinon. However, many unanswered questions still exist regarding the environmental fate and degradation mechanisms of this pesticide. The catalytic mechanisms responsible for enzymatic degradation remain unexplained, and ecotechnological techniques need to be applied to gain a comprehensive understanding of these issues. Hence, this review article provides in-depth information about the impact and toxicity of diazinon in living systems and discusses the developed ecotechnological remedial methods used for the effective biodegradation of diazinon in a contaminated environment.

Trimethylamine-N-Oxide Pathway: A Potential Target for the Treatment of MAFLD.

Trimethylamine-N-oxide (TMAO) is a molecular metabolite derived from the gut flora, which has recently emerged as a candidate risk factor for metabolic dysfunction-associated fatty liver disease (MAFLD). TMAO is mainly derived from gut, where the gut microbiota converts TMA precursors into TMA, which is absorbed into the bloodstream through the intestinal mucosa, and then transformed into TMAO by hepatic flavin monooxygenases (FMOs) in the liver. High-nutrient diets rich in TMA precursors, such as red meat, eggs, and fish, are the main sources of TMAO. Excessively consuming such diets not only directly affects energy metabolism in liver, but also increases the concentration of TMAO in plasma, which promotes the development of MAFLD by affecting bile acid metabolism, unfolded protein response, and oxidative stress. In this review, we focused on the relationship between TMAO and MAFLD and summarized intervention strategies for reducing circulating TMAO concentration, aiming at providing new targets for the prevention and treatment of MAFLD.

The flavin monooxygenase Bs3 triggers cell death in plants, impairs growth in yeast and produces H2O2 in vitro.

The pepper resistance gene Bs3 triggers a hypersensitive response (HR) upon transcriptional activation by the corresponding effector protein AvrBs3 from the bacterial pathogen Xanthomonas. Expression of Bs3 in yeast inhibited proliferation, demonstrating that Bs3 function is not restricted to the plant kingdom. The Bs3 sequence shows striking similarity to flavin monooxygenases (FMOs), an FAD- and NADPH-containing enzyme class that is known for the oxygenation of a wide range of substrates and their potential to produce H2O2. Since H2O2 is a hallmark metabolite in plant immunity, we analyzed the role of H2O2 during Bs3 HR. We purified recombinant Bs3 protein from E. coli and confirmed the FMO function of Bs3 with FAD binding and NADPH oxidase activity in vitro. Translational fusion of Bs3 to the redox reporter roGFP2 indicated that the Bs3-dependent HR induces an increase of the intracellular oxidation state in planta. To test if the NADPH oxidation and putative H2O2 production of Bs3 is sufficient to induce HR, we adapted previous studies which have uncovered mutations in the NADPH binding site of FMOs that result in higher NADPH oxidase activity. In vitro studies demonstrated that recombinant Bs3S211A protein has twofold higher NADPH oxidase activity than wildtype Bs3. Translational fusions to roGFP2 showed that Bs3S211A also increased the intracellular oxidation state in planta. Interestingly, while the mutant derivative Bs3S211A had an increase in NADPH oxidase capacity, it did not trigger HR in planta, ultimately revealing that H2O2 produced by Bs3 on its own is not sufficient to trigger HR.

Impact of diet-derived trimethylamine N-oxide (TMAO) on endothelial dysfunction: An in vitro study

Background and Aims: Trimethylamine N-oxide (TMAO) is an organic compound directly absorbed from food products or endogenously synthesized from its precursors, choline and L-carnitine, that are metabolized in trimethylamine by the gut microbiota and then oxidized in TMAO by hepatic flavin monooxygenases. At physiological concentrations TMAO acts as an osmolyte, a protein stabilizer and an electron acceptor. Emerging studies show an association between high TMAO circulating levels and atherosclerotic damage. Purpose of the study is to evaluate the direct effect of TMAO on bovine aortic endothelial cells (BAEC) key parameters related to vascular dysfunction.

Facile and label-free fluorescence strategy for evaluating the influence of bioactive ingredients on FMO3 activity via supramolecular host-guest reporter pair

Screening inhibitors of flavin monooxygenase 3 (FMO3) is very important for treating trimethylamine N-oxide (TMAO) derived thrombotic diseases. Herein, focusing on Xuefu Zhuyu decoction (XFZYD) as a Chinese traditional medicine with antithrombotic efficacy, a facile and label-free fluorescence strategy was developed for evaluating the influence of the bioactive ingredients in XFZYD on FMO3 activity through indicator displacement assay. To this end, the optimized supramolecular host-guest (p-sulfonatocalix[4]arene-oxazine 1) reporter pair and FMO3 catalytic system were exploited to determine the influence of the bioactive compounds in XFZYD on the conversion from TMA to TMAO. From the nine compounds tested, naringin, paeoniflorin, β-ecdysterone, 18β-glycyrrhizic acid, amygdalin, albiflorin, and saikosaponin A downregulated FMO3 activity and reduced TMAO biosynthesis. Moreover, molecular docking was successfully applied to simulate the optimal conformation of a receptor-ligand complex between FMO3 and all tested compounds except for β-ecdysterone. Therefore, this approach provides a novel and promising strategy for screening FMO3 inhibitors from Chinese traditional medicine by supramolecular sensing.