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Riboflavin-5-phosphate (riboflavin) is the most commonly used photosensitizer in corneal crosslinking (CXL); while its efficient delivery into the stroma through the corneal epithelial barrier is challenging. In this paper, we presented novel responsive porous microneedles with ocular microinjection capability to deliver riboflavin controllably inside the cornea to facilitate CXL. The microneedle patch was composed of Poly (N-isopropyl acrylamide) (PNIPAM), graphene oxide (GO), and riboflavin-loaded gelatin. After penetrating the cornea by the stiff and porous gelatin needle tip, the photothermal-responsive characteristic of the PNIPAM/GO hydrogel middle layer could realize the contraction of the gel under the stimulation of near-infrared light, which subsequently could control the release of riboflavin from the backing layer into the cornea stromal site both in vitro and in vivo. Based on the microneedles system, we have demonstrated that this microinjection technique exhibited superior riboflavin delivery capacity and treatment efficacy to the conventional epithelial-on protocol in a rabbit keratoconus model, with benefits including minimal invasiveness and precise administering. Thus, we believe the responsive porous microneedles with riboflavin ocular microinjection capability are promising for clinical corneal crosslinking without epithelial debridement. [ABSTRACT FROM AUTHOR]

Root morphology, an important determinant of nutrient absorption and plant growth, can adapt to various growth environments to promote survival. Solution flow under hydroponic conditions provides a mechanical stimulus, triggering adaptive biological responses, including altered root morphology and enhanced root growth and surface area to facilitate nutrient absorption. To clarify these mechanisms, we applied untargeted metabolomics technology, detecting 1737 substances in lettuce root samples under different flow rates, including 17 common differential metabolites. The abscisic acid metabolic pathway product dihydrophaseic acid and the amino and nucleotide sugar metabolism factor N-acetyl-d-mannosamine suggest that nutrient solution flow rate affects root organic acid and sugar metabolism to regulate root growth. Spatial metabolomics analysis of the most stressed root bases revealed significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways: "biosynthesis of cofactors" and "amino sugar and nucleotide sugar metabolism". Colocalization analysis of pathway metabolites revealed a flow-dependent spatial distribution, with higher flavin mononucleotide, adenosine-5′-diphosphate, hydrogenobyrinic acid, and D-glucosamine 6-phosphate under flow conditions, the latter two showing downstream-side enrichment. In contrast, phosphoenolpyruvate, 1-phospho-alpha-D-galacturonic acid, 3-hydroxyanthranilic acid, and N-acetyl-D-galactosamine were more abundant under no-flow conditions, with the latter two concentrated on the upstream side. As metabolite distribution is associated with function, observing their spatial distribution in the basal roots will provide a more comprehensive understanding of how metabolites influence plant morphology and response to environmental changes than what is currently available in the literature. [ABSTRACT FROM AUTHOR]

Despite the increasing demand for efficient and sustainable chemical processes, the development of scalable systems using biocatalysis for fine chemical production remains a significant challenge. We have developed a scalable flow system using immobilized enzymes to facilitate flavin-dependent biocatalysis, targeting as a proof-of-concept asymmetric alkene reduction. The system integrates a flavin-dependent Old Yellow Enzyme (OYE) and a soluble hydrogenase to enable H2-driven regeneration of the OYE cofactor FMNH2. Molecular hydrogen was produced by water electrolysis using a proton exchange membrane (PEM) electrolyzer and introduced into the flow system via a designed gas membrane addition module at a high diffusion rate. The flow system shows remarkable stability and reusability, consistently achieving >99% conversion of ketoisophorone to levodione. It also demonstrates versatility and selectivity in reducing various cyclic enones and can be extended to further flavin-based biocatalytic approaches and gas-dependent reactions. This electro-driven continuous flow system, therefore, has significant potential for advancing sustainable processes in fine chemical synthesis. Flavin-based biocatalysis using flavin mononucleotide (FMN) cofactor attracts significant attention for its application in asymmetric alkene reduction and various other reactions, however, the scale-up of flavin-based biocatalysis in flow remains unexplored. Here, the authors develop a closed-loop flow platform for H2-driven regeneration of cofactor FMNH2 and ene-reduction using immobilized Old Yellow Enzyme, achieving >99% conversion of ketoisophorone to levodione. [ABSTRACT FROM AUTHOR]

AbstractEne-reductase (ER) has been widely applied for asymmetrical synthesis of chiral intermediates due to its substrate promiscuity, photoexcited reactivity, and excellent property with producing two chiral centers at a time. Natural ERs often exhibit the same stereoselectivity, and they need to be engineered for opposite configuration of chiral compounds. The hydrogenation process toward activated alkenes by ERs is composed of reductive half reaction and oxidative half reaction, which are dependent upon two cofactors NAD(P)H and flavin mononucleotide. The catalytic activity of ERs will be affected by the size of the substrate, the activating strength of the electron-withdrawing groups, redox potential of cofactors, and the loop flexibility around catalytic cavity. Currently, protein engineering to ERs has been successfully employed to enhance various catalytic properties, including photoexcited asymmetric synthesis. This review summarizes the approaches to reverse the stereoselectivity and enhance catalytic activity of ERs and new applications of the engineered ERs in photobiocatalytic asymmetric synthesis, besides the discussion with the existing molecular mechanisms of mutants regarding the improved catalytic performance. [ABSTRACT FROM AUTHOR]

Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae (Kp) is an infectious disease pathogen that poses a significant global health threat due to its potential to cause severe infections and its tendency to exhibit multidrug resistance. Understanding the enzymatic mechanisms of the oxygen‐insensitive nitroreductases (Kp‐NRs) from Kp is crucial for the development of effective nitrofuran drugs, such as nitrofurantoin, that can be activated as antibiotics. In this paper, three crystal structures of two Kp‐NRs (PDB entries 7tmf/7tmg and 8dor) are presented, and an analysis of their crystal structures and their flavin mononucleotide (FMN)‐binding mode is provided. The structures with PDB codes 7tmf (Kp‐NR1a), 7tmg (Kp‐NR1b) and 8dor (Kp‐NR2) were determined at resolutions of 1.97, 1.90 and 1.35 Å, respectively. The Kp‐NR1a and Kp‐NR1b structures adopt an αβ fold, in which four‐stranded antiparallel β‐sheets are surrounded by five helices. With domain swapping, the β‐sheet was expanded with a β‐strand from the other molecule of the dimer. The difference between the structures lies in the loop spanning Leu173–Ala185: in Kp‐NR1a the loop is disordered, whereas the loop adopts multiple conformations in Kp‐NR1b. The FMN interactions within Kp‐NR1/NR2 involve hydrogen‐bond and π‐stacking interactions. Kp‐NR2 contains four‐stranded antiparallel β‐sheets surrounded by eight helices with two short helices and one β‐sheet. Structural and sequence alignments show that Kp‐NR1a/b and Kp‐NR2 are homologs of the Escherichia coli oxygen‐insensitive NRs YdjA and NfnB and of Enterobacter cloacae NR, respectively. By homology inference from E. coli, Kp‐NR1a/b and Kp‐NR2 may detoxify polynitroaromatic compounds and Kp‐NR2 may activate nitrofuran drugs to cause bactericidal activity through a ping‐pong bi‐bi mechanism, respectively. [ABSTRACT FROM AUTHOR]

Incidence of esophageal and gastric cancer has been linked to low B‐vitamin status. We conducted matched nested case–control studies of incident esophageal squamous cell carcinoma (ESCC; 340 case–control pairs) and gastric cancer (GC; 352 case–control pairs) within the Golestan Cohort Study. The primary exposure was plasma biomarkers: riboflavin and flavin mononucleotide (FMN) (vitamin B2), pyridoxal phosphate (PLP) (B6), cobalamin (B12), para‐aminobenzoylglutamate (pABG) (folate), and total homocysteine (tHcy); and indicators for deficiency: 3‐hydroxykyurenine‐ratio (HK‐r for vitamin B6) and methylmalonic acid (MMA for B12). We estimated odds ratios (ORs) and 95% confidence intervals (CIs) using conditional logistic regression adjusting for matching factors and potential confounders. High proportions of participants had low B‐vitamin and high tHcy levels. None of the measured vitamin B levels was associated with the risk of ESCC and GC, but elevated level of MMA was marginally associated with ESCC (OR = 1.42, 95% CI = 0.99–2.04) and associated with GC (OR = 1.53, 95% CI = 1.05–2.22). Risk of GC was higher for the highest versus lowest quartile of HK‐r (OR = 1.95, 95%CI = 1.19–3.21) and for elevated versus non‐elevated HK‐r level (OR = 1.59, 95% CI = 1.13–2.25). Risk of ESCC (OR = 2.81, 95% CI = 1.54–5.13) and gastric cancer (OR = 2.09, 95%CI = 1.17–3.73) was higher for the highest versus lowest quartile of tHcy. In conclusion, insufficient vitamin B12 was associated with higher risk of ESCC and GC, and insufficient vitamin B6 status was associated with higher risk of GC in this population with prevalent low plasma B‐vitamin status. Higher level of tHcy, a global indicator of OCM function, was associated with higher risk of ESCC and GC. [ABSTRACT FROM AUTHOR]

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. Methods: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). Results: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M− 1.S− 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M− 1.S− 1. Conclusions: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes. [ABSTRACT FROM AUTHOR]

Uracil-thymine dehydrogenase (UTDH), which catalyzes the irreversible oxidation of uracil to barbituric acid in oxidative pyrimidine metabolism, was purified from Rhodococcus erythropolis JCM 3132. The finding of unusual stabilizing conditions (pH 11, in the presence of NADP+ or NADPH) enabled the enzyme purification. The purified enzyme was a heteromer consisting of three different subunits. The enzyme catalyzed oxidation of uracil to barbituric acid with artificial electron acceptors such as methylene blue, phenazine methosulfate, benzoquinone, and α-naphthoquinone; however, NAD+, NADP+, flavin adenine dinucleotide, and flavin mononucleotide did not serve as electron acceptors. The enzyme acted not only on uracil and thymine but also on 5-halogen-substituted uracil and hydroxypyrimidine (pyrimidone), while dihydropyrimidine, which is an intermediate in reductive pyrimidine metabolism, and purine did not serve as substrates. The activity of UTDH was enhanced by cerium ions, and this activation was observed with all combinations of substrates and electron acceptors. • Uracil-thymine dehydrogenase was found to be a heteromer consisting of three different subunits. • Uracil-thymine dehydrogenase catalyzed oxidation of uracil with artificial electron acceptors. • The activity of uracil-thymine dehydrogenase was enhanced by cerium ions. [ABSTRACT FROM AUTHOR]

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress. [ABSTRACT FROM AUTHOR]

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterolgrown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4- diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate. [ABSTRACT FROM AUTHOR]

Flavin mononucleotide (FMN)‐dependent ene‐reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon–carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene‐reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH‐preferring 12‐oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene‐reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β‐hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β‐hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β‐hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β‐hairpin flap favors NADH binding. [ABSTRACT FROM AUTHOR]

Enzymes reliant on pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5′-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms. [ABSTRACT FROM AUTHOR]

The presence of high chromium (Cr) levels induces the buildup of reactive oxygen species (ROS), resulting in hindered plant development. Riboflavin (vitamin B2) is produced by plants, fungi, and microbes. It serves as a precursor to the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which play a crucial role in cellular metabolism. The objective of this work was to clarify the underlying mechanisms by which riboflavin alleviates Cr stress in Praecitrullus fistulosus L. Further, the role of riboflavin in growth, ions homeostasis, methylglyoxal detoxification, and antioxidant defense mechanism are not well documented in plants under Cr toxicity. We found greater biomass and minimal production of ROS in plants pretreated with riboflavin under Cr stress. Results manifested a clear abridge in growth, chlorophyll content, and nutrient uptake in Indian squash plants exposed to Cr stress. Findings displayed that Cr stress visibly enhanced oxidative injury reflected as higher malondialdehyde (MDA), hydrogen peroxide (H2O2), superoxide radical (O2•‒), methylglyoxal (MG) levels alongside vivid lipoxygenase activity. Riboflavin strengthened antioxidant system, enhanced osmolyte production and improved membrane integrity. Riboflavin diminished Cr accumulation in aerial parts that led to improved nutrient acquisition. Taken together, riboflavin abridged Cr phytotoxic effects by improving redox balance because plants treated with riboflavin had strong antioxidant system that carried out effective ROS detoxification. Riboflavin protected membrane integrity that, in turn, improved nutrient uptake in plants. [ABSTRACT FROM AUTHOR]

Despite the crucial role of peroxisomes in cellular redox maintenance, little is known about how these organelles transport redox metabolites across their membrane. In this study, we sought to assess potential associations between the cellular redox landscape and the human peroxisomal solute carrier SLC25A17, also known as PMP34. This carrier has been reported to function as a counter-exchanger of adenine-containing cofactors such as coenzyme A (CoA), dephospho-CoA, flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD+), adenosine 3′,5′-diphosphate, flavin mononucleotide, and adenosine monophosphate. We found that inactivation of SLC25A17 resulted in a shift toward a more reductive state in the glutathione redox couple (GSSG/GSH) across HEK-293 cells, HeLa cells, and SV40-transformed mouse embryonic fibroblasts, with variable impact on the NADPH levels and the NAD+/NADH redox couple. This phenotype could be rescued by the expression of Candida boidinii Pmp47, a putative SLC25A17 orthologue reported to be essential for the metabolism of medium-chain fatty acids in yeast peroxisomes. In addition, we provide evidence that the alterations in the redox state are not caused by changes in peroxisomal antioxidant enzyme expression, catalase activity, H 2 O 2 membrane permeability, or mitochondrial fitness. Furthermore, treating control and Δ SLC25A17 cells with dehydroepiandrosterone, a commonly used glucose-6-phosphate dehydrogenase inhibitor affecting NADPH regeneration, revealed a kinetic disconnection between the peroxisomal and cytosolic glutathione pools. Additionally, these experiments underscored the impact of SLC25A17 loss on peroxisomal NADPH metabolism. The relevance of these findings is discussed in the context of the still ambiguous substrate specificity of SLC25A17 and the recent observation that the mammalian peroxisomal membrane is readily permeable to both GSH and GSSG. [Display omitted] • Loss of SLC25A17 selectively lowers the overall peroxisomal redox state. • Loss of SLC25A17 impacts peroxisomal NADPH metabolism. • The peroxisomal and cytosolic glutathione pools are kinetically disconnected. [ABSTRACT FROM AUTHOR]

Flavin mononucleotide (FMN) produces photo-induced reactive oxygen species (ROS), making it a bio-based and sustainable photosensitizer for micropollutant degradation. However, the rapid self-degradation of FMN under light poses challenges in practical applications. We propose for the first time to use porous organic polymer (POP) structures as particles and in situ grown on nanofibrous membranes to capture the ribityl side chain ("tail") of FMN by electrostatic-driven guest-host interaction. By restraining the free bending mode of FMN in POP, its self-degradation is highly inhibited, showing a prolonged half-life (102.7 and 79.7 times to that in solution and in β-cyclodextrin, respectively) without any impact on the ROS production even after 16 h of UVA irradiation. As a proof-of-concept, the photocatalytic degradation efficiency of FMN-POP complexes can be achieved at 58-93% against micropollutants under UVA. The stabilization of FMN by the "tail" capture in the POP allows its photocatalytic degradation function to be continuously online.

Background: Pickled mustard, the largest cultivated vegetable in China, generates substantial waste annually, leading to significant environmental pollution due to challenges in timely disposal, leading to decomposition and sewage issues. Consequently, the imperative to address this concern centers on the reduction and comprehensive resource utilization of raw mustard waste (RMW). To achieve complete and quantitative resource utilization of RMW, this study employs novel technology integration for optimizing its higher-value applications. Results: Initially, subcritical hydrothermal technology was applied for rapid decomposition, with subsequent ammonia nitrogen removal via zeolite. Thereafter, photosynthetic bacteria, Rhodopseudomonas palustris, were employed to maximize hydrogen and methane gas production using various fermentation enhancement agents. Subsequent solid-liquid separation yielded liquid fertilizer from the fermented liquid and soil amendment from solid fermentation remnants. Results indicate that the highest glucose yield (29.6 ± 0.14) was achieved at 165–173℃, with a total sugar content of 50.2 g/L and 64% glucose proportion. Optimal ammonia nitrogen removal occurred with 8 g/L zeolite and strain stable growth at 32℃, with the highest OD600 reaching 2.7. Several fermentation promoters, including FeSO4, Neutral red, Na2S, flavin mononucleotide, Nickel titanate, Nickel oxide, and Mixture C, were evaluated for hydrogen production. Notably, Mixture C resulted in the maximum hydrogen production (756 mL), a production rate of 14 mL/h, and a 5-day stable hydrogen production period. Composting experiments enhanced humic acid content and organic matter (OM) by 17% and 15%, respectively. Conclusions: This innovative technology not only expedites RMW treatment and hydrogen yield but also substantially enriches soil fertility. Consequently, it offers a novel approach for low-carbon, zero-pollution RMW management. The study's double outcomes extend to large-scale RMW treatment based on the aim of full quantitative resource utilization of RMW. Our method provides a valuable reference for waste management in similar perishable vegetable plantations. [ABSTRACT FROM AUTHOR]

Antibiotic resistance remains a pressing global concern, with most antibiotics targeting the bacterial ribosome or a limited range of proteins. One class of underexplored antibiotic targets is bacterial riboswitches, structured RNA elements that regulate key biosynthetic pathways by binding a specific ligand. We developed a methodology termed Fluorescent Ligand Equilibrium Displacement (FLED) to rapidly discover small molecules that bind the flavin mononucleotide (FMN) riboswitch. FLED leverages intrinsically fluorescent FMN and the quenching effect on RNA binding to create a label-free, in vitro method to identify compounds that can bind the apo population of riboswitch in a system at equilibrium. The response difference between known riboswitch ligands and controls demonstrates the robustness of the method for high-throughput screening. An existing drug discovery library that was screened using FLED resulted in a final hit rate of 0.67%. The concentration response of each hit was determined and revealed a variety of approximate effective concentration values. Our preliminary screening data support the use of FLED to identify small molecules for medicinal chemistry development as FMN riboswitch-targeted antibiotic compounds. This robust, label-free, and cell-free method offers a strong alternative to other riboswitch screening methods and can be adapted to a variety of laboratory setups. [ABSTRACT FROM AUTHOR]

Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5–8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. Key points: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv [ABSTRACT FROM AUTHOR]

Background: Methylene blue (MB) and flavin mononucleotide (FMN)-mediated photodynamic therapy (PDT) have demonstrated local antimicrobial effect, but no direct comparative study has been published so far for the treatment of toenail onychomycosis. Objectives: To directly compare the short and medium-term efficacy of MB versus FMN as photosensitizers in PDT for toenail onychomycosis by applying them in a 40% w/w urea cream in two different dye concentrations. Methods: Forty toenails with distal and lateral subungual moderate onychomycosis due to dermatophyte fungi were randomised to receive 10 weekly sessions of PDT mediated by four topical formulations including MB or FMN at two different concentrations: Group I: 0.1% w/w MB; Group II: 2% w/w MB; Group III: 0.1% w/w FMN; and Group IV: 2% w/w FMN. Photographs were used for onychomycosis severity index (OSI) estimation allowing clinical assessment at any point of the study. Microscopic and microbiological evaluations were carried out at baseline, 27- and 35-week followups. Side effects were recorded along with patient satisfaction. Results: At week 27, mycological cure rates were 60%, 30%, 50% and 40% and complete cure rates were 0%, 20%, 10% and 20%, for Groups I, II, III and IV respectively. At week 35, mycological cure rates were 70%, 70%, 70% and 60% and complete cure rates were 30%, 50%, 70% and 30%, for Groups I, II, III and IV respectively. All cream formulations were safe and patients were fairly satisfied. Conclusions: Results of the present work confirm PDT as a therapeutic alternative for onychomycosis. Although all cream formulations were safe and effective, with a good degree of satisfaction, higher cure rates were obtained with 2% w/w MB cream and 0.1% w/w FMN cream. [ABSTRACT FROM AUTHOR]

In the previously published results, we studied the effect of flavin mononucleotide (FMN), sodium halides NaF and NaBr, iron chloride FeCl3 and aluminum chloride AlCl3 on zigzag patterns of films obtained from saline solutions of bovine serum albumin (BSA). Changes in the properties of the biopolymer and its aqueous environment in solutions were monitored by UV absorption and fluorescence spectroscopy, microwave dielectrometry and dynamic light scattering. This work summarizes the obtained results on the influence of components of various nature on BSA and its film textures. Changes in the parameters of zigzag patterns are associated with polydispersity due to the presence of colloidal particles, aggregates and FMN associates, as well as a decrease in the surface potential and BSA hydration. The possibility of predicting the character of the influence of the studied biologically active substances on BSA is analyzed. [ABSTRACT FROM AUTHOR]

Phenolic off-flavor compounds such as 4-vinylguaiacol (4-VG) affect the quality of fermented foods. In Saccharomyces cerevisiae, 4-VG production requires prenylated FMN (flavin mononucleotide) produced by the mitochondrial protein Pad1. To examine this mechanism further, we expressed Escherichia coli ubiX, whose gene product prenylates FMN in E. coli, in an S. cerevisiae Δpad1 strain, which recovered the ability to produce 4-VG. Because E. coli UbiX is expected to localize to the cytoplasm in the transformed yeast, next, we expressed a truncated Pad1 (tPad1) variant lacking the mitochondrial translocation signal sequence in S. cerevisiae Δpad1. The transformed strain also recovered the ability to produce 4-VG; furthermore, it produced a larger amount of 4-VG than wild-type S. cerevisiae, indicating that 4-VG production in S. cerevisiae may be mediated by the partial mislocalization of Pad1 to the cytoplasm. We also expressed the gene encoding Dekkera (Brettanomyces) bruxellensis phenolic acid decarboxylase (DbPAD) in S. cerevisiae Δpad1. The transformed yeast recovered the ability to produce 4-VG, indicating that the D. bruxellensis phenolic acid decarboxylation reaction does not require prenylated FMN. Sequencing of the cloned DbPAD gene showed that the translation start codon differs from previous reports; therefore, we recloned the DbPAD gene from the position predicted to be the translation initiation point (designated DbPADs). Transformation of S. cerevisiae Δpad1 with DbPADs further increased the amount of 4-VG produced. Collectively, these findings provide insight into the mechanism of off-flavor production by different yeast species. [ABSTRACT FROM AUTHOR]

Flavin mononucleotide (FMN) in methanol was photochemically reduced to FMNH2, which undergoes electron transfer with Methyl viologen (MV2+) in ethanol. This charge transfer interaction results in the reduction of MV2+ into MV+˙, as well as the storage of electrons. The spectroscopic experiments allow for the elucidation of quantitative electron transfer into MV2+. To study the redox potential for FMN species, the HOMO and LUMO energies were calculated using density functional theory (DFT) method on the B3LYP level with a basis set of 6-311G(d,p). The HOMO and LUMO energy calculations show that the redox state of FMNH2 is energetically more favorable than FMNH˙ for MV2+ reduction. These studies revealed good agreement between the experimental results and computational predictions. Moreover, molecular electrostatic potential (MEP) was performed on the FMNH2-MV2+ complex to determine the site of electron transfer, and the results were compared to the experimental results. FMN functionalized to the surface of colloidal titanium dioxide nanoparticles (TiO2 NPs) showed enhanced reactivity toward the reduction of perfluorooctanoic acid (PFOA). The two-electron transfer reduction process is generated by irradiating FMN attached to TiO2, during which FMN reduces to FMNH2. The FMNH2 then reacts with PFOA to produce shorter chain structures with fewer fluorine atoms, such as C5F11COOH. The results indicate that the rational organization of FMNH2/TiO2 to mediate two-electron transfer is effective for the reductive degradation and remediation of PFOA. [ABSTRACT FROM AUTHOR]

Fusaric acid (FA) is a mycotoxin produced by several Fusarium species. Burkholderia ambifaria T16 is a rhizosphere bacterium, able to use FA as sole nitrogen, carbon, and energy source. By screening a transposon insertional library, combined with proteomic analysis, genes and enzymes involved in the microbial degradation of FA were identified for the first time. A functional 2-methylcitrate cycle, an anaplerotic pathway where propionyl-coenzyme A (CoA) is converted to pyruvate and succinate, was shown to be essential for growth in the presence of FA. The proteomic profile of B. ambifaria T16 showed that more than 50 enzymes (including those belonging to the 2-methylcitrate cycle, fatty acid metabolism, valine catabolism, and flavin biosynthesis) were significantly more abundant when growing on FA than on citrate. Flavin mononucleotide (FMN)- dependent luciferases like monooxygenase (LLM) are shown to catalyze the pyridine-ring cleavage reaction of several N-heterocyclic compounds. Deletion of a gene encoding a predicted LLM enzyme that was highly upregulated during growth on FA, completely abolished the capability of B. ambifaria T16 to grow with this mycotoxin as sole nitrogen, carbon, and energy source. Re-introduction of the wild type gene was able to restore growth. The mentioned gene is part of a gene cluster of unknown function that we termed fua, due to its probable role in fusaric acid catabolism. Our results suggest that the LLM encoded in the fua cluster catalyzes the pyridine-ring opening reaction during FA degradation, and that propionyl-CoA is one of the intermediates of FA catabolism in B. ambifaria T16. IMPORTANCE Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium. [ABSTRACT FROM AUTHOR]

Flavin mononucleotide (FMN) is a dye belonging to the flavin family. These dyes produce photosensitized degradation of organic compounds via reaction with the excited states of the dye or with reactive oxygen species photogenerated from the triplet of the dye. This article presents a new polymeric dye (FMN–CS) composed of the photosensitizer FMN covalently bonded to chitosan polysaccharide (CS). FMN–CS obtained has a molecular weight of 230 × 103 g mol−1 and a deacetylation degree of 74.8%. The polymeric dye is an environmentally friendly polymer with spectroscopic and physicochemical properties similar to those of FMN and CS, respectively. Moreover, under sunlight, it is capable of generating 1O2 with a quantum yield of 0.31. FMN–CS, like CS, is insoluble in basic media. This allows easy recovery of the polymeric dye once the photosensitized process has been carried out and makes FMN–CS a suitable photosensitizer for the degradation of pollutants in contaminated waters. To evaluate whether FMN–CS may be used for pollutant degradation, the photosensitized degradation of two trihydroxybenzenes by FMN–CS was studied. [ABSTRACT FROM AUTHOR]

Visible light-induced photocrosslinking techniques have attracted significant attention for their flexibility, controllability, safety, and energy conservation, especially in tissue engineering and biofabrication, compared to UV photocrosslinking. Despite these advantages, current photoinitiators are constrained by various challenges, including inadequate photoinitiation efficiency, low biocompatibility, poor water solubility, and limited compatibility with diverse crosslinking systems. Here, a water-soluble derivative of riboflavin, flavin mononucleotide (FMN−), was used to assess its potential as an initiator in multiple-photocrosslinking systems, including radical photopolymerization, dityrosine, and ditryptophan coupling crosslinking, under blue light irradiation. Blue light irradiation facilitated an efficient electron transfer reaction between FMN− and persulfate, owing to their suitable spectral compatibility and photoactivity. The resulting oxidizing free radicals and excited triplet state of FMN− served as initiating active species for the multiple-photocrosslinking reactions. The combination of FMN− and potassium persulfate (KPS) exhibited exceptional photoinitiation efficiency for various biomaterials, including silk fibroin, gelatin, poly(ethylene glycol) diacrylate, and carboxymethyl cellulose modified with amino acids. Furthermore, the cytocompatibility of the FMN−/KPS photoinitiator was demonstrated by the survival rates of 3T3-LI fibroblasts encapsulated in it, which exceeded 95 % when compared to a commercial initiator. By introducing persulfate, the photoinitiation efficiency of flavin mononucleotide was significantly improved. The application scenarios of flavin mononucleotide and persulfate combinations were also greatly extended, including radical photopolymerization, dityrosine, diphenylalanine, and ditryptophan coupling crosslinking. Among them, the coupling crosslinking of amino acids (di-phenylalanine, and di-tryptophan) modified carboxymethyl cellulose, to our knowledge, was first reported. The excellent cytocompatibility of cell encapsulation further proved that the combinations of flavin mononucleotide and persulfate have great potential in tissue engineering. [Display omitted] [ABSTRACT FROM AUTHOR]

Simple Summary: Riboflavin, an essential B-vitamin, plays a crucial role in poultry metabolism, impacting energy production, growth, and immune regulation. Its role in redox reactions and energy metabolism is vital for optimal growth and development. Riboflavin is essential for ATP production and the conversion of tryptophan into niacin. Deficiency can lead to skeletal deformities, impaired growth, and compromised immune function. Dietary riboflavin supplementation is necessary due to variable bioavailability in plant-derived sources. The vitamin is absorbed through specialized transport proteins, and its cellular uptake is facilitated by specific receptors. Riboflavin's role in protein synthesis and its antioxidant properties influence poultry growth and defense against oxidative stress. Its impact on reproductive performance, hatchability, and overall poultry health underscores its significance in poultry nutrition. Future research should focus on its interactions with other nutrients, exploring analogs, and integrating advanced technologies like precision nutrition and nanotechnology for enhanced delivery. Riboflavin, or vitamin B2, is indispensable for poultry, profoundly impacting their metabolic equilibrium, growth, and overall health. In a climate of increasing demand for poultry products and heightened production intensity, grasping the multifaceted roles of riboflavin in domestic fowl nutrition becomes paramount. This essential vitamin serves as a precursor to two vital coenzymes, flavin mononucleotide and flavin adenine dinucleotide, integral players in pivotal redox reactions and energy metabolism. Inadequate riboflavin levels translate into stunted growth, skeletal deformities, and compromised feed conversion efficiency, thereby adversely affecting poultry performance and bottom-line profitability. Riboflavin goes beyond its fundamental role, ameliorating nutrient utilization, facilitating protein synthesis, and augmenting enzyme activity, rightfully earning its epithet as the "growth-promoting vitamin". Poultry's reproductive success intricately hinges on riboflavin levels, dictating egg production and hatchability. It is imperative to note that riboflavin requirements exhibit variations among poultry species and distinct production phases, emphasizing the importance of judicious and balanced supplementation strategies. Aligning dietary recommendations with genetic advancements holds the promise of fostering sustainable growth within the poultry sector. Exploring the multifaceted aspects of riboflavin empowers researchers, nutritionists, and producers to elevate poultry nutrition and overall well-being, harmonizing with the industry's evolving demands. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) of proteins are paramount in health and disease. Phosphoproteome analysis by enrichment techniques is becoming increasingly attractive for biomedical research. Recent findings show co-enrichment of other phosphate-containing biologically relevant PTMs, but these results were obtained by closed searches focused on the modifications sought. Open searches are a breakthrough in high-throughput PTM analysis (OS-PTM), identifying practically all PTMs detectable by mass spectrometry, even unknown ones, with their modified sites, in a hypothesis-free and deep manner. Here we reanalyze liver phosphoproteome by OS-PTM, demonstrating its extremely complex nature. We found extensive Lys glycerophosphorylations (pgK), as well as modification with glycerylphosphorylethanolamine on Glu (gpetE) and flavin mononucleotide on His (fmnH). The functionality of these metabolite-derived PTMs is demonstrated during metabolic dysfunction-associated steatotic liver disease (MASLD) development in mice. MASLD elicits specific alterations in pgK, epgE and fmnH in the liver, mainly on glycolytic enzymes and mitochondrial proteins, suggesting an increase in glycolysis and mitochondrial ATP production from the early insulin-resistant stages. Thus, we show new possible mechanisms based on metabolite-derived PTMs leading to intrahepatic lipid accumulation during MASLD development and reinforce phosphoproteome enrichment as a valuable tool with which to study the functional implications of a variety of low-abundant phosphate-containing PTMs in cell physiology. [ABSTRACT FROM AUTHOR]

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

The spectroscopy and photo-induced dissociation of flavin mononucleotide anions in vacuo are investigated over the 300–500 nm wavelength range. Comparison of the dependence of fragment ion yields as a function of deposited photon energy with calculated dissociation energies and collision-induced dissociation measurements performed under single-collision conditions suggests that a substantial fraction of photo-activated ions decompose through non-statistical fragmentation pathways. Among these pathways is the dominant photo-induced fragmentation channel, the loss of a fragment identified as formylmethylflavin. The fragment ion specific action spectra reveal electronic transition energies close to those for flavins in solution and previously published gas-phase measurements, although the photo-fragment yield upon excitation of the S2 ← S0 transition appears to be suppressed. [ABSTRACT FROM AUTHOR]

Summary: Background: To report on a concept of liver assessment during ex situ hypothermic oxygenated perfusion (HOPE) and its significant impact on liver utilization. Methods: An analysis of prospectively collected data on donation after circulatory death (DCD) livers, treated by HOPE at our institution, during a 11-year period between January 2012 and December 2022. Findings: Four hundred and fifteen DCD Maastricht III livers were offered during the study period in Switzerland, resulting in 249 liver transplants. Of those, we performed 158 DCD III liver transplants at our institution, with 1-year patient survival and death censored graft survival (death with functioning graft) of 87 and 89%, respectively, thus comparable to benchmark graft survivals of ideal DBD and DCD liver transplants (89% and 86%). Correspondingly, graft loss for primary non-function or cholangiopathy was overall low, i.e., 7/158 (4.4%) and 11/158 (6.9%), despite more than 82% of DCD liver grafts ranked high (6–10 points) or futile risk (>10 points) according to the UK-DCD score. Consistently, death censored graft survival was not different between low-, high-risk or futile DCD III livers. The key behind these achievements was the careful development and implementation of a routine perfusate assessment of mitochondrial biomarkers for injury and function, i.e., release of flavin mononucleotide from complex I, perfusate NADH, and mitochondrial CO2 production during HOPE, allowing a more objective interpretation of liver quality on a subcellular level, compared to donor derived data. Interpretation: HOPE after cold storage is a highly suitable and easy to perform perfusion approach, which allows reliable liver graft assessment, enabling surgeons to make a fact based decision on whether or not to implant the organ. HOPE-treatment should be combined with viability assessment particularly when used for high-risk organs, including DCD livers or organs with relevant steatosis. Funding: This study was supported by the Swiss National Foundation (SNF) grant 320030_189055/1 to PD.

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5‐deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one‐electron‐transfer reactions in flavoproteins are impeded with 5‐deazaflavin as the cofactor. Based on these findings, it was concluded that the 5‐deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re‐oxidizes or undergoes disproportionation. The long‐standing paradigm of 5‐deazaflavin being solely a two‐electron/hydride acceptor/donor—"a nicotinamide in flavin clothing"—needs to be re‐evaluated now with the indirect observation of a one‐electron‐reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo‐CIDNP) 1H nuclear magnetic resonance (NMR) under biologically relevant conditions. [ABSTRACT FROM AUTHOR]

During indigo dyeing fermentation, indigo reduction for the solubilization of indigo particles occurs through the action of microbiota under anaerobic alkaline conditions. The original microbiota in the raw material (sukumo: composted indigo plant) should be appropriately converged toward the extracellular electron transfer (EET)-occurring microbiota by adjusting environmental factors for indigo reduction. The convergence mechanisms of microbiota, microbial physiological basis for indigo reduction, and microbiota led by different velocities in the decrease in redox potential (ORP) at different fermentation scales were analyzed. A rapid ORP decrease was realized in the big batch, excluding Actinomycetota effectively and dominating Alkalibacterium, which largely contributed to the effective indigo reduction. Functional analyses of the microbiota related to strong indigo reduction on approximately day 30 indicated that the carbohydrate metabolism, prokaryotic defense system, and gene regulatory functions are important. Because the major constituent in the big batch was Alkalibacterium pelagium, we attempted to identify genes related to EET in its genome. Each set of genes for flavin adenine dinucleotide (FAD) transportation to modify the flavin mononucleotide (FMN)-associated family, electron transfer from NADH to the FMN-associated family, and demethylmenaquinone (DMK) synthesis were identified in the genome sequence. The correlation between indigo intensity reduction and metabolic functions suggests that V/A-type H+/Na+-transporting ATPase and NAD(P)H-producing enzymes drive membrane transportations and energization in the EET system, respectively. [ABSTRACT FROM AUTHOR]

While aqueous organic redox flow batteries (RFBs) represent potential solutions to large-scale grid storage, their electrolytes suffer from short lifetimes due to rapid degradation. We show how an understanding of these degradation processes can be used to dramatically improve performance, as illustrated here via a detailed study of the redox-active biomolecule, flavin mononucleotide (FMN), a molecule readily derived from vitamin B2. Via in-situ nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) we identify FMN hydrolysis products and show that these give rise to the additional plateau seen during charging of an FMN-cyanoferrate battery. The redox reactions of the hydrolysis product are not reversible, but we demonstrate that capacity is still retained even after substantial hydrolysis, albeit with reduced voltaic efficiency, FMN acting as a redox mediator. Critically, we demonstrate that degradation is mitigated and battery efficiency is substantially improved by lowering the pH to 11. Furthermore, the addition of cheap electrolyte salts to tune the pH results in a dramatic increase in solubility (above 1 M), this systematic improvement of the flavin-based system bringing RFBs one step closer to commercial viability. Aqueous organic redox-flow batteries are known to suffer capacity loss via degradation of the redox-active species. Here, the authors use in situ methods to study the electrolyte flavin mononucleotide, identifying a redox mediator mechanism that mitigates capacity loss and a route to prevent its degradation. [ABSTRACT FROM AUTHOR]

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes. [ABSTRACT FROM AUTHOR]

Biomarkers are powerful clinical diagnostics and predictors of patient outcome. However, robust measurements often require time and expensive laboratory equipment, which is insufficient to track rapid changes and limits direct use in the operating room. Here, this study presents a portable spectrophotometric device for continuous real‐time measurements of fluorescent and non‐fluorescent biomarkers at the point of care. This study measures the mitochondrial damage biomarker flavin mononucleotide (FMN) in 26 extended criteria human liver grafts undergoing hypothermic oxygenated perfusion to guide clinical graft assessment. Real‐time data identified seven organs unsuitable for transplant that are discarded. The remaining grafts are transplanted and FMN values correlated with post‐transplant indicators of liver function and patient recovery. Further, this study shows how this device can be used to monitor dialysis patients by measuring creatinine in real‐time. Our approach provides a simple method to monitor biomarkers directly within biological fluids to improve organ assessment, patient care, and biomarker discovery. [ABSTRACT FROM AUTHOR]

Metal-based electromagnetic interference shielding materials are limited in their application due to their susceptibility to corrosion. They are therefore being replaced by corrosion-resistant polymers and carbon nanotubes. However, it is difficult to disperse carbon nanotubes within polymer matrices as they aggregate because of van der Waals forces. Therefore, to improve the dispersion of carbon nanotubes, we modified thin-wall carbon nanotubes with flavin mononucleotides and tested them by manufacturing a polymer composite. Nylon 12 was chosen as the polymer matrix owing to its excellent abrasion resistance and low wettability. Furthermore, upon annealing the modified carbon nanotubes, the tensile strength of the composite and dispersion after modification increased. When 10 wt% of the annealed modified nanotubes was used, the composite exhibited a sheet resistance of 8.7 Ω/sq and an EMI shielding effectiveness of 29.4 dB in the X-band. The novel composite can be advantageous for use in electronic equipment due to its low weight and flexibility. [ABSTRACT FROM AUTHOR]

High serum uric acid levels, known as hyperuricemia (HUA), are associated with an increased risk of developing gout, chronic kidney disease, cardiovascular disease, diabetes, and other metabolic syndromes. In this study, a promising bacterial strain capable of biodegrading uric acid (UA) was successfully isolated from Baijiu cellar mud using UA as the sole carbon and energy source. The bacterial strain was identified as Bacillus paramycoides-YC02 through 16S rDNA sequence analysis. Under optimal culture conditions at an initial pH of 7.0 and 38 °C, YC02 completely biodegraded an initial UA concentration of 500 mg/L within 48 h. Furthermore, cell-free extracts of YC02 were found to catalyze and remove UA. These results demonstrate the strong biodegradation ability of YC02 toward UA. To gain further insight into the mechanisms underlying UA biodegradation by YC02, the draft genome of YC02 was sequenced using Illumina HiSeq. Subsequent analysis revealed the presence of gene1779 and gene2008, which encode for riboflavin kinase, flavin mononucleotide adenylyl transferase, and flavin adenine dinucleotide (FAD)-dependent urate hydroxylase. This annotation was based on GO or the KEEG database. These enzymes play a crucial role in the metabolism pathway, converting vitamin B2 to FAD and subsequently converting UA to 5-hydroxyisourate (HIU) with the assistance of FAD. Notably, HIU undergoes a slow non-enzymatic breakdown into 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and (S)-allantoin. The findings of this study provide valuable insights into the metabolism pathway of UA biodegradation by B. paramycoides-YC02 and offer a potential avenue for the development of bacterioactive drugs against HUA and gout. [ABSTRACT FROM AUTHOR]

Fusaric acid (FA) is a well-known mycotoxin that plays an important role in plant pathology. The biosynthetic gene cluster for FA has been identified, but the biosynthetic pathway remains unclarified. Here, we elucidated the biosynthesis of FA, which features a two-enzyme catalytic cascade, a pyridoxal 5-phosphate (PLP)-dependent enzyme (Fub7), and a flavin mononucleotide (FMN)-dependent oxidase (Fub9) in synthesizing the picolinic acid scaffold. FA biosynthesis also involves an off-line collaboration between a highly reducing polyketide synthase (HRPKS, Fub1) and a nonribosomal peptide synthetase (NRPS)-like carboxylic acid reductase (Fub8) in making an aliphatic α,β-unsaturated aldehyde. By harnessing the stereoselective C-C bond-forming activity of Fub7, we established a chemoenzymatic route for stereoconvergent synthesis of a series of 5-alkyl-, 5,5-dialkyl-, and 5,5,6-trialkyl-l-pipecolic acids of high diastereomeric ratio.

Enzymes reliant on pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5′-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B6 vitamers. However, for those depending on vitamin B6 as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.

In this contribution, the relative hyperfine couplings are determined for the 1H nuclei of the flavin mononucleotide (FMN) radical in an aqueous environment. In addition, three structural analogs with different methylation patterns are characterized and the influence of the substituents at the isoalloxazine moiety on the electronic structure of the radicals is explored. By exploiting nuclear hyperpolarization generated via the photo-CIDNP (chemically induced dynamic nuclear polarization) effect, it is possible to study the short-lived radical species generated by in situ light excitation. Experimental data are extracted by least-squares fitting and supported by quantum chemical calculations and published values from electron paramagnetic resonance and electron-nuclear double resonance. Furthermore, mechanistic details of the photoreaction of the investigated flavin analogs with l-tryptophan are derived from the photo-CIDNP spectra recorded at different pH values. Thereby, the neutral and anionic radicals of FMN and three structural analogs are, for the first time, characterized in terms of their electronic structure in an aqueous environment. [ABSTRACT FROM AUTHOR]

Due to their biological importance, the photochemistry of blue-light photoreceptor proteins has been studied extensively over the last few decades. Most blue-light photoreceptors, such as cryptochromes and phototropins, utilize flavin chromophores as their cofactors. After irradiation with light, the chromophore undergoes electron transfer with nearby redox-active amino-acid residues within the protein, whereby this first step of signal transduction may be initiated either from the flavin's excited singlet or triplet state. Despite the collective effort of theoreticians and experimentalists to characterize and understand the photochemistry of flavoproteins, the mechanistic details of the excited state processes initiating signal transduction are yet to be revealed. Here, we use a light-oxygen-voltage-sensing domain from Avena sativa phototropin to get additional insight into the excited state photochemistry of flavoproteins. The influence of structural variations of the cofactor flavin mononucleotide (FMN) is explored by varying the methyl substitution pattern in positions 7 and 8 of the flavin core. The photophysical properties of the FMN derivatives, in the absence and presence of the protein environment, are investigated by UV-vis absorption, fluorescence, and electron paramagnetic resonance spectroscopies as well as cyclic voltammetry. The comparison of the properties of the modified flavin cofactors with those of FMN shows that the rates of the different excited state reactions, and therefore also the singlet/triplet yields, can be modulated substantially by only minor structural modifications of the flavin core. [ABSTRACT FROM AUTHOR]

Photo-CIDNP is one among different promising techniques to improve nuclear magnetic resonance (NMR) sensitivity. Being sensitive to particular amino acids makes it a promising candidate with respect to biological NMR. Our goal is to exploit new sensitizers and compare them to the present working horses flavin mononucleotide, bipyridyl, and the recently applied fluorescein. Among the investigated dyes, we found a new and very efficient one, Atto Thio 12, conveniently used in fluorescence microscopy with 2–3 fold enhanced polarization when compared to the established ones. We also show that diode lasers which are now available from near UV to IR can be efficiently used in photo-CIDNP. Results are obtained with the amino acids tyrosine and tryptophan. [ABSTRACT FROM AUTHOR]

Background: Actinomycetes Streptomyces davaonensis and Streptomyces cinnabarinus synthesize a promising broad-spectrum antibiotic roseoflavin, with its synthesis starting from flavin mononucleotide and proceeding through an immediate precursor, aminoriboflavin, that also has antibiotic properties. Roseoflavin accumulation by the natural producers is rather low, whereas aminoriboflavin accumulation is negligible. Yeasts have many advantages as biotechnological producers relative to bacteria, however, no recombinant producers of bacterial antibiotics in yeasts are known. Results: Roseoflavin biosynthesis genes have been expressed in riboflavin- or FMN-overproducing yeast strains of Candida famata and Komagataella phaffii. Both these strains accumulated aminoriboflavin, whereas only the latter produced roseoflavin. Aminoriboflavin isolated from the culture liquid of C. famata strain inhibited the growth of Staphylococcus aureus (including MRSA) and Listeria monocytogenes. Maximal accumulation of aminoriboflavin in shake-flasks reached 1.5 mg L− 1 (C. famata), and that of roseoflavin was 5 mg L− 1 (K. phaffii). Accumulation of aminoriboflavin and roseoflavin by K. phaffii recombinant strain in a bioreactor reached 22 and 130 mg L− 1, respectively. For comparison, recombinant strains of the native bacterial producer S. davaonensis accumulated near one-order less of roseoflavin while no recombinant producers of aminoriboflavin was reported at all. Conclusions: Yeast recombinant producers of bacterial antibiotics aminoriboflavin and roseoflavin were constructed and evaluated. [ABSTRACT FROM AUTHOR]

The genus Rimicaris is the dominant organism living in hydrothermal vents. However, little research has been done on the functions of their intestinal flora. Here, we investigated the potential functions of Deferribacterota, which is dominant in the intestine of Rimicaris kairei from the Central Indian Ridge. In total, six metagenome-assembled genomes (MAGs) of Deferribacterota were obtained using the metagenomic approach. The six Deferribacterota MAGs (Def-MAGs) were clustered into a new branch in the phylogenetic tree. The six Def-MAGs were further classified into three species, including one new order and two new genera, based on the results of phylogenetic analysis, relative evolutionary divergence (RED), average nucleotide identity (ANI), average amino acid identity (AAI) and DNA–DNA hybridization (DDH) values. The results of the energy metabolism study showed that these bacteria can use a variety of carbon sources, such as glycogen, sucrose, salicin, arbutin, glucose, cellobiose, and maltose. These bacteria have a type II secretion system and effector proteins that can transport some intracellular toxins to the extracellular compartment and a type V CRISPR–Cas system that can defend against various invasions. In addition, cofactors such as biotin, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) synthesized by R. kairei gut Deferribacterota may also assist their host in surviving under extreme conditions. Taken together, the potential function of Deferribacterota in the hydrothermal R. kairei gut suggests its long-term coevolution with the host. [ABSTRACT FROM AUTHOR]

Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p<0.0001). This increase was similar for both groups (p = 0.83). No correlation, however, was found between FI(ex450;em500-600) and post-transplant outcomes, including day 5 or 7 serum creatinine (p = 0.11; p = 0.16), immediate graft function (p = 0.91), creatinine clearance and biopsy-proven rejection at one year (p = 0.14; p = 0.59). LC-MS/MS validation experiments of samples detected FMN in only one perfusate sample, whilst the majority of samples with the highest fluorescence (n = 37/38, 97.4%) remained negative. In the context of clinical kidney HMP, fluorescence spectroscopy unfortunately appears to be not specific and probably unsuitable for FMN. This study shows that FMN does not classify as a clinically relevant predictive biomarker of kidney graft function after transplantation. [ABSTRACT FROM AUTHOR]

The recent outbreak of the COVID-19 pandemic caused by severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2) has shown the necessity for fast and broad drug discovery methods to enable us to react quickly to novel and highly infectious diseases. A well-known SARS-CoV-2 target is the viral main 3-chymotrypsin-like cysteine protease (Mpro), known to control coronavirus replication, which is essential for the viral life cycle. Here, we applied an interaction-based drug repositioning algorithm on all protein-compound complexes available in the protein database (PDB) to identify Mpro inhibitors and potential novel compound scaffolds against SARS-CoV-2. The screen revealed a heterogeneous set of 692 potential Mpro inhibitors containing known ones such as Dasatinib, Amodiaquine, and Flavin mononucleotide, as well as so far untested chemical scaffolds. In a follow-up evaluation, we used publicly available data published almost two years after the screen to validate our results. In total, we are able to validate 17% of the top 100 predictions with publicly available data and can furthermore show that predicted compounds do cover scaffolds that are yet not associated with Mpro. Finally, we detected a potentially important binding pattern consisting of 3 hydrogen bonds with hydrogen donors of an oxyanion hole within the active side of Mpro. Overall, these results give hope that we will be better prepared for future pandemics and that drug development will become more efficient in the upcoming years. [ABSTRACT FROM AUTHOR]