Search

The generation of the active form of vitamin B-6, pyridoxal 5'-phosphate (PLP), in tissues is dependent upon riboflavin as flavin mononucleotide, but whether this interaction is important for maintaining vitamin B-6 status is unclear.To investigate vitamin B-6 and riboflavin status, their metabolic interaction, and relationship with methylenetetrahydrofolate reductase (MTHFR) genotype in adulthood.Data from 5612 adults aged 18-102 y were drawn from the Irish National Adult Nutrition Survey (NANS; population-based sample) and the Trinity-Ulster Department of Agriculture (TUDA) and Genovit cohorts (volunteer samples). Plasma PLP and erythrocyte glutathione reductase activation coefficient (EGRac), as a functional indicator of riboflavin, were determined.Older (≥65 y) compared with younger (65 y) adults had significantly lower PLP concentrations (P 0.001). A stepwise decrease in plasma PLP was observed across riboflavin categories, from optimal (EGRac ≤1.26), to suboptimal (EGRac: 1.27-1.39), to deficient (EGRac ≥1.40) status, an effect most pronounced in older adults (mean ± SEM: 76.4 ± 0.9 vs 65.0 ± 1.1 vs 55.4 ± 1.2 nmol/L; P 0.001). In individuals with the variant MTHFR 677TT genotype combined with riboflavin deficiency, compared with non-TT (CC/CT) genotype participants with sufficient riboflavin, we observed PLP concentrations of 52.1 ± 2.9 compared with 76.8 ±0.7 nmol/L (P 0.001). In participants with available dietary data (i.e., NANS cohort, n = 936), PLP was associated with vitamin B-6 intake (nonstandardized regression coefficient β: 2.49; 95% CI 1.75, 3.24; P 0.001), supplement use (β: 81.72; 95% CI: 66.01, 97.43; P 0.001), fortified food (β: 12.49; 95% CI: 2.08, 22.91; P = 0.019), and EGRac (β: -65.81; 95% CI: -99.08, -32.54; P 0.001), along with BMI (β: -1.81; 95% CI: -3.31, -0.30; P = 0.019).These results are consistent with the known metabolic dependency of PLP on flavin mononucleotide (FMN) and suggest that riboflavin may be the limiting nutrient for maintaining vitamin B-6 status, particularly in individuals with the MTHFR 677TT genotype. Randomized trials are necessary to investigate the PLP response to riboflavin intervention within the dietary range. The TUDA study and the NANS are registered at www.ClinicalTrials.gov as NCT02664584 (27 January 2016) and NCT03374748 (15 December 2017), respectively.Clinical Trial Registry details: Trinity-Ulster-Department of Agriculture (TUDA) study, ClinicalTrials.gov no. NCT02664584 (January 27th 2016); National Adult Nutrition Survey (NANS), ClinicalTrials.gov no. NCT03374748 (December 15th 2017).

Abstract Sequence-based screening has been widely applied in the discovery of novel microbial enzymes. However, majority of the sequences in the genomic databases were annotated using computational approaches and lacks experimental characterization. Hence, the success in obtaining the functional biocatalysts with improved characteristics requires an efficient screening method that considers a wide array of factors. Recombinant expression of microbial enzymes is often hampered by the undesirable formation of inclusion body. Here, we present a systematic in silico screening method to identify the proteins expressible in soluble form and with the desired biological properties. The screening approach was adopted in the recombinant expression of dimethyl sulfide (DMS) monooxygenase in Escherichia coli. DMS monooxygenase, a two-component enzyme consisting of DmoA and DmoB subunits, was used as a model protein. The success rate of producing soluble and active DmoA is 71% (5 out of 7 genes). Interestingly, the soluble recombinant DmoA enzymes exhibited the NADH:FMN oxidoreductase activity in the absence of DmoB (second subunit), and the cofactor FMN, suggesting that DmoA is also an oxidoreductase. DmoA originated from Janthinobacterium sp. AD80 showed the maximum NADH oxidation activity (maximum reaction rate: 6.6 µM/min; specific activity: 133 µM/min/mg). This novel finding may allow DmoA to be used as an oxidoreductase biocatalyst for various industrial applications. The in silico gene screening methodology established from this study can increase the success rate of producing soluble and functional enzymes while avoiding the laborious trial and error involved in the screening of a large pool of genes available. Key points • A systematic gene screening method was demonstrated. • DmoA is also an oxidoreductase capable of oxidizing NADH and reducing FMN. • DmoA oxidizes NADH in the absence of external FMN.

Many healthcare workers who handle anticancer drugs are at risk for occupational exposure. However, there are no established permissible limits for occupational exposure to anticancer drugs; thus, in this study, we aimed to search for and improve procedures that have a greater impact on the amount of spatter for handling anticancer drugs in vials, which are frequently used, based on the quantitative evaluation of the amount of exposure. We used sodium riboflavin phosphate (FMN) as a simulated anticancer drug and measured the amount of FMN dispersed to the handling area by the wiping method and the amount of FMN dispersed in both gloves using high-performance liquid chromatography with fluorescence detection (HPLC-FL). In this study, it was suggested that the overall amount of dispersal in the preparation process was affected by the different methods of injecting the drug solution into the infusion bottles and whether recapping. It was also found that the variation in the amount of dispersal differed depending on the selected preparation technique. It was suggested that the amount of dispersal could be reduced by selecting an appropriate dissolution method for multiple vials, recapping, an appropriate method for injecting the drug into the infusion bottle, and properly preparing the internal pressure of the infusion bottle. The results of this study suggest that there are some techniques and procedures in the preparation process of vials that have a significant effect on the amount of dispersal, and that proper implementation of these techniques can contribute to the reduction of dispersal.

The ability of the human malaria parasite Plasmodium falciparum to access and utilize vital nutrients is critical to its growth and proliferation. Molecules that interfere with these processes could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilization of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focusing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4 to 5 days. Our data are consistent with riboflavin metabolism and/or the utilization of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

Lumichrome (LC) is the major photodegradation product of biologically important flavin cofactors. Since LC serves as a structural comparison with the flavins; understanding excited states of LC is fundamentally important to establish a connection with photophysics of different flavins, such as lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Herein, we deduce the initial excited state structural dynamics of LC using UV resonance Raman (UVRR) intensity analysis. The UVRR spectra at wavelengths across the 260 nm absorption band of LC were measured and resulting Raman excitation profiles and absorption spectrum were self-consistently simulated using a time-dependent wave packet formalism to extract the initial excited state structural and solvent broadening parameters. These results are compared with those obtained for other flavins following UV excitations. We find that LC undergoes a very distinct instantaneous charge redistribution than flavins, which is attributed to the extended π-conjugation present in flavins but missing in LC. The homogeneous broadening linewidth of LC appears to be lower than that of LF, while the inhomogeneous broadening values are comparable, indicating greater solvent interaction with excited flavin on ultrafast timescale compared with LC, whereas on longer timescale these interactions are almost similar.

Consensus sequences have the potential to help classify the structure and function of proteins and highlight key regions that may contribute to their biological properties. Often, the level of significance will track with the extent of sequence conservation, but this should not be considered universal. Arg and Lys dominate a position adjacent to the N1 and C2 carbonyl of flavin mononucleotide (FMN) bound in the proteins of the nitroreductase superfamily. Although this placement satisfies expectations for stabilizing the reduced form of FMN, the substitution of these residues in three subfamilies promoting distinct reactions demonstrates their importance to catalysis as only modest. Replacing Arg34 with Lys, Gln, or Glu enhances FMN binding to a flavin destructase (BluB) by twofold and diminishes FMN turnover by no more than 25%. Similarly, replacing Lys14 with Arg, Gln, or Glu in a nitroreductase (NfsB) does not perturb the binding of the substrate nitrofurazone. The catalytic efficiency does decrease by 21-fold for the K14Q variant, but no change in the midpoint potential of FMN was observed with any of the variants. Equivalent substitution at Arg38 in iodotyrosine deiodinase (IYD) affects catalysis even more modestly (10-fold). While the Arg/Lys to Glu substitution inactivates NfsB and IYD, this change also stabilizes one-electron transfer in IYD contrary to predictions based on other classes of flavoproteins. Accordingly, functional correlations developed in certain structural superfamilies may not necessarily translate well to other superfamilies.

miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen. Graphical abstract

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.

RNA molecules play critical roles in cellular functions at the level of gene expression and regulation. The intricate 3D structures and the functional roles of RNAs make RNA molecules ideal targets for therapeutic drugs. The rational design of RNA-targeted drug requires accurate modeling of RNA-ligand interactions. Recently a new computational tool, RLDOCK, was developed to predict ligand binding sites and binding poses. Using an iterative multiscale sampling and search algorithm and a energy-based evaluation of ligand poses, the method enables efficient and accurate predictions for RNA-ligand interactions. Here we present a detailed illustration of the computational procedure for the practical implementation of the RLDOCK method. Using Flavin mononucleotide (FMN) docking to F. nucleatum FMN riboswitch as an example, we illustrate the computational protocol for RLDOCK-based prediction of RNA- ligand interactions. The RLDOCK software is freely accessible at http://https://github.com/Vfold-RNA/RLDOCK.

Serum FMN, instead of riboflavin, was inversely associated with colorectal cancer risk in the Chinese population, especially among individuals with lower serum PLP and higher serum folate levels.

Photoactivities and photoinduced antibacterial functions of two vitamin B2 (VB2) derivatives, riboflavin (RF) and flavin mononucleotide (FMN), were investigated by computational modeling and variou...

Self-resistance genes are deployed by many microbial producers of bioactive natural products to avoid self-toxicity. Myxin, a di-N-oxide phenazine produced by Lysobacter antibioticus OH13, is toxic to many microorganisms and tumor cells. Here, we uncovered a self-defense strategy featuring the antibiotic biosynthesis monooxygenase (ABM) family protein LaPhzX for myxin degradation. The gene LaPhzX is located in the myxin biosynthetic gene cluster (LaPhz), and its deletion resulted in bacterial mutants that are more sensitive to myxin. In addition, the LaPhzX mutants showed increased myxin accumulation and reduction of its derivative, compound 4, compared to the wild-type strain. Meanwhile, in vitro biochemical assays demonstrated that LaPhzX significantly degraded myxin in the presence of nicotinamide adenine dinucleotide phosphate (NADPH), nicotinamide adenine dinucleotide (NADH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). In addition, heterologous expression of LaPhzX in Xanthomonas oryzae pv. oryzae and Escherichia coli increased their resistance to myxin. Overall, our work illustrates a monooxygenase-mediated self-resistance mechanism for phenazine antibiotic biosynthesis.

Riboflavin is an essential nutrient for humans and animals, and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in the cells. Therefore, riboflavin and its derivatives are widely used in the food, pharmaceutical, nutraceutical and cosmetic industries. Advances in biotechnology have led to a complete shift in the commercial production of riboflavin from chemical synthesis to microbial fermentation. In this review, we provide a comprehensive review of biotechnologies that enhance riboflavin production in microorganisms, as well as representative examples. Firstly, the synthesis pathways and metabolic regulatory processes of riboflavin in microorganisms; and the current strategies and methods of metabolic engineering for riboflavin production are systematically summarized and compared. Secondly, the using of systematic metabolic engineering strategies to enhance riboflavin production is discussed, including laboratory evolution, histological analysis and high-throughput screening. Finally, the challenges for efficient microbial production of riboflavin and the strategies to overcome these challenges are prospected.

Flavin mono-nucleotides (FMNs) are cofactors that hold responsibility for carrying and transferring electrons in the electron transport chain stage of cellular respiration. Without being facilitated by FMNs, energy production is stagnant due to the interruption in most of the cellular processes. Investigation on FMN's functions, therefore, can gain holistic understanding about human diseases and molecular information on drug targets. We proposed a deep learning model using a two-dimensional convolutional neural network and position specific scoring matrices that could identify FMN interacting residues with the sensitivity of 83.7 percent, specificity of 99.2 percent, accuracy of 98.2 percent, and Matthews correlation coefficients of 0.85 for an independent dataset containing 141 FMN binding sites and 1,920 non-FMN binding sites. The proposed method outperformed other previous studies using similar evaluation metrics. Our positive outcome can also promote the utilization of deep learning in dealing with various problems in bioinformatics and computational biology.

Flavin-binding fluorescent proteins are promising geneti-cally encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even be-tween different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy. Generation and optical characterization of CagFbFP variants was supported by the Russian Science Foundation, grant number 21-64-00018. A. B. acknowledges the support of the Ministry of Science and Higher Education of the Russian Federation (agreement 075-03-2023-106, project FSMG-2020-0003) for fluorescence microscopy experiments.

A study on the kinetics of the reaction between cyanocobalamin (vitamin B12, CNCbl) and the reduced form of flavin mononucleotide (FMNH2) in weakly acidic, neutral, and slightly alkaline media is performed. It is shown that FMNH2 can reduce CNCbl to cobalamin(II) (Cbl(II)). It is established that protonated, mono-deprotonated, and di-deprotonated forms of FMNH2 can participate in the reaction. The reaction mechanism includes the slow substitution of the 5,6-dimethylbenzimidazole nucleotide with FMNH2 molecule, rapid electron transfer from FMNH2 to Co(III) ions, and the subsequent dissociation of cyanide. The reaction is reversible, due to the ability of the oxidized form of FMNH2 (FMN) to react with the Cbl(II) complex.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.

Nitric oxide synthase (NOS) is a homodimeric flavohemoprotein responsible for catalyzing the oxidation of l-arginine (l-Arg) to citrulline and nitric oxide. Electrons are supplied for the reaction via interdomain electron transfer between an N-terminal heme-containing oxygenase domain and a FMN-containing (sub)domain of a C-terminal reductase domain. Extensive attention has focused on elucidating how conformational dynamics regulate electron transfer between the domains. Here we investigate the impact of the interdomain FMN-heme interaction on the heme active site dynamics of inducible NOS (iNOS). Steady state linear and time-resolved two-dimensional infrared (2D IR) spectroscopy was applied to probe a CO ligand at the heme within the oxygenase domain for full-length and truncated or mutated constructs of human iNOS. Whereas the linear IR spectra of the CO ligand were identical among the constructs, 2D IR spectroscopy revealed variation in the frequency dynamics. The wild-type constructs that can properly form the FMN/oxygenase docked state due to the presence of both the FMN and oxygenase domains showed slower dynamics than the oxygenase domain alone. Introduction of the mutation (E546N) predicted to perturb electrostatic interactions between the domains resulted in measured dynamics intermediate between those for the full-length and individual oxygenase domain, consistent with perturbation to the docked/undocked equilibrium. These results indicate that docking of the FMN domain to the oxygenase domain not only brings the FMN cofactor within electron transfer distance of the heme domain but also modulates the dynamics sensed by the CO ligand within the active site in a way expected to promote efficient electron transfer.

The Lyme disease agent, Borrelia burgdorferi, harbors a significantly reduced genome and relies on the scavenging of critical nutrients from its tick and mammalian hosts for survival. Riboflavin salvage has been shown to be important for B. burgdorferi infection of mice, yet the contributions of riboflavin to B. burgdorferi metabolism and survival in the tick remain unknown. Using a targeted mass spectrometry approach, we confirmed the importance of bb0318, the putative ATPase component of an ABC-type riboflavin transporter, for riboflavin salvage and the production of FMN and FAD. This analysis further revealed that Δbb0318 B. burgdorferi displayed increased levels of glycerol 3-phosphate compared to the wild-type. The glycerol 3-phosphate dehydrogenase activity of GlpD was found to be FAD-dependent and the transcription and translation of glpD were significantly decreased in Δbb0318 B. burgdorferi. Finally, gene bb0318 was found to be important for maximal spirochete burden in unfed larvae and essential for survival in feeding ticks. Together, these data demonstrate the importance of riboflavin salvage for B. burgdorferi carbon metabolism and survival in ticks.

l-Lactate oxidase (LOx) is a flavin mononucleotide (FMN)-dependent triose phosphate isomerase (TIM) barrel fold enzyme that catalyzes the oxidation of l-lactate using oxygen as a primary electron acceptor. Although reductive half-reaction mechanism of LOx has been studied by structure-based kinetic studies, oxidative half-reaction and substrate/product-inhibition mechanisms were yet to be elucidated. In this study, the structure and enzymatic properties of wild-type and mutant LOxs from Enterococcus hirae (EhLOx) were investigated. EhLOx structure showed the common TIM-barrel fold with flexible loop region. Noteworthy observations were that the EhLOx crystal structures prepared by co-crystallization with product, pyruvate, revealed the complex structures with "d-lactate form ligand," which was covalently bonded with a Tyr211 side chain. This observation provided direct evidence to suggest the product-inhibition mode of EhLOx. Moreover, this structure also revealed a flip motion of Met207 side chain, which is located on the flexible loop region as well as Tyr211. Through a saturation mutagenesis study of Met207, one of the mutants Met207Leu showed the drastically decreased oxidase activity but maintained dye-mediated dehydrogenase activity. The structure analysis of EhLOx Met207Leu revealed the absence of flipping in the vicinity of FMN, unlike the wild-type Met207 side chain. Together with the simulation of the oxygen-accessible channel prediction, Met207 may play as an oxygen gatekeeper residue, which contributes oxygen uptake from external enzyme to FMN. Three clades of LOxs are proposed based on the difference of the Met207 position and they have different oxygen migration pathway from external enzyme to active center FMN.

Background Riboflavin is a precursor of FMN and FAD which act as coenzymes of numerous enzymes. Riboflavin is an important biotechnological commodity with annual market sales exceeding nine billion US dollars. It is used primarily as a component of feed premixes, a food colorant, a component of multivitamin mixtures and medicines. Currently, industrial riboflavin production uses the bacterium, Bacillus subtilis, and the filamentous fungus, Ashbya gossypii, and utilizes glucose and/or oils as carbon substrates. Results We studied riboflavin biosynthesis in the flavinogenic yeast Candida famata that is a genetically stable riboflavin overproducer. Here it was found that the wild type C. famata is characterized by robust growth on lactose and cheese whey and the engineered strains also overproduce riboflavin on whey. The riboflavin synthesis on whey was close to that obtained on glucose. To further enhance riboflavin production on whey, the gene of the transcription activator SEF1 was expressed under control of the lactose-induced promoter of the native β-galactosidase gene LAC4. These transformants produced elevated amounts of riboflavin on lactose and especially on whey. The strain with additional overexpression of gene RIB6 involved in conversion of ribulose-5-phosphate to riboflavin precursor had the highest titer of accumulated riboflavin in flasks during cultivation on whey. Activation of riboflavin synthesis was also obtained after overexpression of the GND1 gene that is involved in the synthesis of the riboflavin precursor ribulose-5-phosphate. The best engineered strains accumulated 2.5 g of riboflavin/L on whey supplemented only with (NH4)2SO4 during batch cultivation in bioreactor with high yield (more than 300 mg/g dry cell weight). The use of concentrated whey inhibited growth of wild-type and engineered strains of C. famata, so the mutants tolerant to concentrated whey were isolated. Conclusions Our data show that the waste of dairy industry is a promising substrate for riboflavin production by C. famata. Possibilities for using the engineered strains of C. famata to produce high-value commodity (riboflavin) from whey are discussed.

Phosphate ion (PO

NTO (5-nitro-1,2,4-triazol-3-one), an energetic material used in military applications, may be released to the environment during manufacturing, transportation, storage, training, and disposal. A detailed investigation of the possible mechanism for all steps of reduction of NTO by oxygen-insensitive nitroreductase, as one of the pathways for NTO environmental degradation, was performed by computational study at the PCM(Pauling)/M06-2X/6-311++G(d,p) level. Obtained results reveal an overall sequence for NTO transformation into ATO (5-amino-1,2,4-triazol-3-one) with the flavin mononucleotide (FMN) cofactor of nitroreductase. Reduction of the nitro group to the nitroso group and the nitroso group to the hydroxylamino group follow a similar mechanism that consists of the sequential electron and proton transfer from the flavin cofactor. The hydride transfer mechanism may contribute to reduction of the nitroso group by the anionic form of the reduced flavin cofactor. Reduction of 5-(hydroxylamino)-1,2,4-triazol-3-one by the neutral form of the reduced flavin is impossible, whereas reduction of the hydroxylamino group to the amino group occurs with the anionic form of the reduced cofactor by a mechanism involving an initial proton transfer from the hydroxonium ion followed by two electrons and one proton transfers from the flavin cofactor. Small activation energies and high exothermicity support the significant contribution of oxygen-insensitive nitroreductase and other enzymes, containing FMN as a cofactor, to NTO degradation in the environment.

Estimating the binding energies of small molecules to RNA could help uncover their molecular recognition characteristics and be used to rationally design RNA-targeting chemical probes. Here, we leveraged the ability of the fragment molecular orbital (FMO) method to provide detailed pairwise energetic information to examine the interactions between the aptamer domain of the flavin mononucleotide (FMN)-responsive riboswitch and small-molecule ligands. After developing an efficient protocol for executing high-level FMO calculations on RNA-ligand complexes, we applied our protocol to nine FMN-aptamer-ligand complexes. We then used the results to identify "hot-spots" within the aptamer and decomposed pairwise interactions between the hot-spot residues and the ligands. Interestingly, we found that several of these hot-spot residues interact with the ligands via atypical CH···O hydrogen bonds and anion-π contacts, as well as (face-to-edge) T-shaped π-π interactions. We envision that our results should pave the way for the wider and more prominent use of FMO calculations to study structure-energy relationships in diverse RNA-ligand systems, which in turn may provide a basis for dissecting the molecular recognition characteristics of RNAs.

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH− to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor.

Redox cofactors mediate many enzymatic processes and are increasingly employed in biomedical and energy applications. Exploring the influence of external magnetic fields on redox cofactor chemistry can enhance our understanding on magnetic field-sensitive biological processes and allow the application of magnetic fields to modulate redox reactions involving cofactors. Through a combination of experiments and modeling, we investigate the influence of magnetic fields on electrochemical reactions in redox cofactor solutions. By employing flavin mononucleotide (FMN) cofactor as a model system, we characterize magnetically induced changes in Faradaic currents. We find that radical pair intermediates have negligible influence on current increases in FMN solution upon application of a magnetic field. The dominant mechanism underlying the observed current increases is the magneto-hydrodynamic effect. We extend our analyses to other diffusion-limited electrochemical reactions of redox cofactor solutions and arrive at similar conclusions, highlighting the opportunity to use this framework in redox cofactor chemistry.

Abstract Materials based on hyaluronic acid (HA) are extensively used in tissue engineering as scaffolds. Photoinduced crosslinking is one way to prepare them, and, for this, HA must be modified with vinyl groups, which are capable of participating in free-radical reactions upon exposure to light. The quantity of grafted vinyl groups, represented as the degree of substitution (DS), is an important parameter of modified HA (mHA) that is related to the mechanical, chemical, and biological properties of scaffolds. Here, we demonstrate the feasibility of tuning DS by varying the reaction parameters (composition and concentration of reaction components and reaction conditions) and investigate the effect of DS on the viscosity of mHA solutions. As example, we consider the photoinduced reaction of mHA in the presence of flavin mononucleotide as the initiator, which can be used in fabrication of noncytotoxic scaffolds by 3D printing. The growth behavior of fibroblasts on the scaffold surface is studied.

TYW1 is a radical S-adenosyl-l-methionine (SAM) enzyme that catalyzes the condensation of pyruvate and N-methylguanosine-containing tRNAPhe, forming 4-demethylwyosine-containing tRNAPhe. Homologues of TYW1 are found in both archaea and eukarya; archaeal homologues consist of a single domain, while eukaryal homologues contain a flavin binding domain in addition to the radical SAM domain shared with archaeal homologues. In this study, TYW1 from Saccharomyces cerevisiae (ScTYW1) was heterologously expressed in Escherichia coli and purified to homogeneity. ScTYW1 is purified with 0.54 ± 0.07 and 4.2 ± 1.9 equiv of flavin mononucleotide (FMN) and iron, respectively, per mole of protein, suggesting the protein is ∼50% replete with Fe-S clusters and FMN. While both NADPH and NADH are sufficient for activity, significantly more product is observed when used in combination with flavin nucleotides. ScTYW1 is the first example of a radical SAM flavoenzyme that is active with NAD(P)H alone.

Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one-electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH(2), but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Viability assessment is one of the main indications for machine perfusion (MP) in liver transplantation. This review summarizes the rationale, evolution and limitations of proposed viability criteria and suggests a framework for future studies.Liver viability is most frequently assessed during normothermic MP by combining parameters relative to perfusate and bile composition, vascular flows and macroscopic aspect. Assessment protocols are largely heterogeneous and have significantly evolved over time, also within the same group, reflecting the ongoing evolution of the subject. Several recent preclinical studies using discarded human livers or animal models have explored other approaches to viability assessment. During hypothermic MP, perfusate flavin mononucleotide has emerged as a promising biomarker of mitochondrial injury and function. Most studies on the subject suffer from limitations, including low numbers, lack of multicenter validation, and subjective interpretation of some viability parameters.MP adds a further element of complexity in the process of assessing the quality of a liver graft. Understanding the physiology of the parameters included in the different assessment protocols is necessary for their correct interpretation. Despite the possibility of assessing liver viability during MP, the importance of donor-recipient matching and operational variables should not be disregarded.

The bacterial nitroreductases (NRs) NfsB and NfsA are conserved homodimeric FMN-dependent flavoproteins that are responsible for the reduction of nitroaromatic substrates. Berberine (BBR) is a plant-derived isoquinoline alkaloid with a large conjugated ring system that is widely used in the treatment of various diseases. It was recently found that the gut microbiota convert BBR into dihydroberberine (dhBBR, the absorbable form) mediated by bacterial NRs. The molecular basis for the transformation of BBR by the gut microbiota remains unclear. Here, kinetic studies showed that NfsB from Escherichia coli (EcNfsB), rather than EcNfsA, is responsible for the conversion of BBR to dhBBR in spite of a low reaction rate. The crystal structure of the EcNfsB–BBR complex showed that BBR binds into the active pocket at the dimer interface, and its large conjugated plane stacks above the plane of the FMN cofactor in a nearly parallel orientation. BBR is mainly stabilized by π-stacking interactions with both neighboring aromatic residues and FMN. Structure-based mutagenesis studies further revealed that the highly conserved Phe70 and Phe199 are important residues for the conversion of BBR. The structure revealed that the C6 atom of BBR (which receives the hydride) is ∼7.5 Å from the N5 atom of FMN (which donates the hydride), which is too distant for hydride transfer. Notably, several well ordered water molecules make hydrogen-bond/van der Waals contacts with the N1 atom of BBR in the active site, which probably donate protons in conjunction with electron transfer from FMN. The structure–function studies revealed the mechanism for the recognition and binding of BBR by bacterial NRs and may help to understand the conversion of BBR by the gut microbiota.

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of

Plasma soluble growth stimulating gene protein 2 (sST2) was rapidly detected through a novel time-resolved fluorescence immunochromatography analysis (TRF-ICA).

Mycophenolate Mofetil (MMF) is an important immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage, however, is limited by severe gastrointestinal toxicity that is observed in approximately 45% of MMF recipients. The active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepatic recirculation by bacterial β-glucuronidase (GUS) enzymes, which reactivate MPA from mycophenolate glucuronide (MPAG) within the gastrointestinal tract. GUS enzymes demonstrate distinct substrate preferences based on their structural features, and gut microbial GUS enzymes that reactivate MPA have not been identified. Here, we compare the fecal microbiomes of transplant recipients receiving MMF to healthy individuals using shotgun metagenomic sequencing. We find that neither microbial composition nor the presence of specific structural classes of GUS genes are sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We next employed a GUS-specific activity-based chemical probe and targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples. The identification of specific GUS enzymes was improved by using the metagenomics data collected from the fecal samples. We found that the presence of GUS enzymes that bind the flavin mononucleotide (FMN) is significantly correlated with efficient MPA reactivation. Furthermore, structural analysis identified motifs unique to these FMN-binding GUS enzymes that provide molecular support for their ability to process this drug glucuronide. These results indicate that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.

Reading is a vision-reliant task, requiring sequential eye movements. Binocularly discordant input results in visual sensory and oculomotor dysfunction in amblyopia, which may contribute to reading difficulties. This study aims to determine the contributions of fixation eye movement (FEM) abnormalities, clinical type and severity of amblyopia to reading performance under binocular and monocular viewing conditions.Twenty-three amblyopic patients and nine healthy controls were recruited. Eye movements elicited during fixation and reading of preselected passages were collected for each subject using infrared video-oculography. Subjects were classified as having no nystagmus (n = 9), fusion maldevelopment nystagmus (FMN, n = 5), or nystagmus without structural anomalies that does not meet criteria for FMN or infantile nystagmus (n = 9). Reading rate (words/min), the number of forward and regressive saccades (per 100 words) and fixation duration (s) were computed.Amblyopic patients with and without nystagmus exhibited greater vergence and fixation instability. In patients without nystagmus, the instability arises from increased amplitude and velocity of fast and slow FEMs respectively. Amblyopic patients with and without nystagmus exhibited lower reading speeds with increased fixation duration, regressive and progressive saccades than controls in all viewing conditions. Mixed etiology, greater amblyopic eye visual acuity and stereopsis deficits were associated with greater reading difficulties under binocular viewing.The presence of oculomotor dysfunction and the extent of visual acuity and stereoacuity deficits contribute to reading difficulties in patients with amblyopia, with and without nystagmus. The understanding of reading difficulties is essential to devise accommodations to limit long-term academic and vocational consequences of amblyopia.

Background: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS). Objective: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD. Methods: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized. Results: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of β-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 μM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 μM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+. Conclusion: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

Here, we demonstrate an interesting strategy of modulating mitochondrial reactive oxygen species (ROS) using the organic electron acceptor molecule carbonyl-bridged bithiazole attached with bis-trifluoroacetophenone (BBT). This molecule was found to affect complex I activity. It has the propensity to bind close to the flavin mononucleotide site of complex I of mitochondria where it traps electron released from nicotinamide adenine dinucleotide (NADH) and elevates intracellular ROS, which suggests that the bridged carbonyl in BBT plays a crucial role in the acceptance of electron from NADH. We understand that the potential of the NADH/NAD+ redox couple and low-lying LUMO energy level of BBT are compatible with each other, thus favoring its entrapment of released electrons in complex I. This effect of BBT in ROS generation activates JNK and p38 stress-dependent pathways and resulted in mitochondrial-dependent apoptotic cell death with the reduction in expression of several important cyto-protecting factors (Hsp27 and NFκB), indicating its potential in inhibition of cancer cell relapse. Intriguingly, we found that BBT is not a P-glycoprotein substrate, which further reveals its excellent anticancer potential. This study enlightens us on how the power of electron acceptor ability became an emerging strategy for modulation of intracellular function.

Flavin adenine dinucleotide synthetase (FADS), a bifunctional prokaryotic enzyme, is involved in the synthesis of two vital cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Here, we investigated the biochemical characteristics of FADS from Staphylococcus aureus (Sa), a pathogenic bacteria causing food-borne diseases. The SaFADS possesses riboflavin kinase (RFK) and FMN adenylyltransferase (FMNAT) activities that transforms riboflavin to FMN and FMN to FAD, respectively. The FMNAT domain also exhibits reversible FAD pyrophosphorylase activity (FADpp). Further, we show that the FMNAT and FADpp activities are dependent on the reducing environment. Mutations of the conserved K289 and F290 residues present on the RFK domain affect the kinetic parameters of both the RFK and FMNAT domains. Additionally, the molecular dynamics analysis of apo and riboflavin: ATP: Mg2+ ternary complex of SaFADS shows that F290 is involved in stabilizing the active site geometry to hold the enzyme-substrate complex. In addition, the deletion of the αh2 helix that acts as a connecting linker between the FMNAT and RFK domains showed substantial loss of their activities. The helix deletion could have affected the flap motion of L2c, L4c, β4n and L3n present in the close proximity resulting in the distortion of the active site geometry. In conclusion, our study has characterized the RFK and FMNAT activities of SaFADS and shown the importance of conserved K289 and F290 in RFK activity. As FADSs are potential drug targets, understanding their mechanism of action might help in discovering species-specific antibacterial drugs.

In vitro, reductive mobilization of ferritin iron using suitable electron transfer mediators has emerged as a possible mechanism to mimic the iron release process, in vivo. Nature uses flavins as electron relay molecules for important biological oxidation and oxygenation reactions. Therefore, the current work utilizes three flavin analogues: riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which differ in size and charge but have similar redox potentials, to relay electron from nicotinamide adenine dinucleotide (NADH) to ferritin mineral core. Of these, the smallest/neutral analogue, RF, released more iron (~ three fold) in comparison to the larger and negatively charged FMN and FAD. Although iron mobilization got marred during the initial stages under aerobic conditions, but increased with a greater slope at the later stages of the reaction kinetics, which gets inhibited by superoxide dismutase, consistent with the generation of O2∙− in situ. The initial step, i.e., interaction of flavins with NADH played critical role in the iron release process. Overall, the flavin-mediated reductive iron mobilization from ferritins occurred via two competitive pathways, involving the reduced form of flavins either alone (anaerobic condition) or in combination with O2∙− intermediate (aerobic condition). Moreover, faster iron release was observed for ferritins from Mycobacterium tuberculosis than from bullfrog, indicating the importance of protein nanocage and the advantages they provide to the respective organisms. Therefore, these structure–reactivity studies of flavins with NADH/O2 holds significance in ferritin iron release, bioenergetics, O2-based cellular toxicity and may be potentially exploited in the treatment of methemoglobinemia. Smaller sized/neutral flavin analogue, riboflavin (RF) exhibits faster reactivity towards both NADH and O2 generating more amount of O2∙− and releases higher amount of iron from different ferritins, compared to its larger sized/negatively charged derivatives such as FMN and FAD.

Flavin mononucleotides (FMNs) and flavin adenine nucleotide (FAD) play vital roles in the electron-transfer processes in diverse enzymatic reactions. Owing to the isoalloxazine chromophore, flavins are easily detectable by surface-enhanced Raman spectroscopy (SERS), a surface-sensitive technique. However, the details of the adsorption of flavins on SERS-active materials have never been investigated. In this study, a comprehensive SERS analysis of flavins containing lumichrome and lumiflavin on silver nanoparticles was conducted. With the aid of density-functional-theory calculations, our results suggested that the flavin molecules were adsorbed on the silver nanoparticles via the N3 site of the isoalloxazine moiety, which had a stronger adsorption ability than the adenine moiety in the FAD. The SERS spectra of the flavins at different pH values also supported this conclusion. This study demonstrated the feasibility of SERS for the structural characterization of flavins, paving the way for the functional exploration of flavin-labeled detection sensors and flavoprotein researches.

Formononetin (FMN) is a methoxy isoflavone found abundantly in leguminous plants and associated foods. Several analytical methods have been developed to detect FMN. However, they are costly, complicated, and time-consuming. This study describes an indirect competitive enzyme-linked immunosorbent assay (icELISA) to determine FMN content in food samples using a monoclonal antibody (mAb) against FMN produced by a newly established hybridoma cell line. Validation studies were conducted, and this assay was found to be sufficiently reliable, with an analytical measurement range of 19.53-1250 ng/mL and a detection limit of 17.42 ng/mL. Furthermore, icELISA was successfully applied for a cell-based assay in which the amount of FMN and ononin uptake was quantified in MC3T3-E1 cells. Hence, icELISA is a simple and reliable method for the detection and quantification of FMN, as well as elucidation of its functions and underlying mechanisms of action.

Lactate sensing has high importance for metabolic diseases diagnostics, food spoilage, sports medicine, or the construction of biofuel cell devices. Therefore, continuous lactate sensing devices which enable accurate detection should be developed. Here we present the overexpression and utilization of FMN-lactate dehydrogenase from Saccharomyces cerevisiae for oxygen-insensitive, continuous amperometric lactate biosensing. The developed sensors exhibit a high signal-to-noise ratio, low interference effect, and a wide range of linear responses using both direct and mediated electron transfer configurations. The thionine-based mediated electron transfer configuration was stable for 8 hours of continuous activity and two weeks of periodic activity with storage at 4°C. We further grafted the redox mediators on multiwall carbon-nanotubes to lower the redox mediator leaching effect. The developed grafting technique improved the biosensor stability and allowed continuous operation for at least 20 hours. Both the mediator-entrapped and the grafted bioanodes were further coupled with a bilirubin oxidase-based biocathode to construct a biofuel cell device. The various biofuel cells have generated a maximal power output of 110µW/cm2 under atmospheric conditions.

Azoreductases are being extensively investigated for their ability to initiate degradation of recalcitrant azo dyes through reduction of azo bonds. There is great interest in studying their diversity, structure, and function to facilitate better understanding and effective application. Current study reports azoreductase enzyme from Bacillus velezensis, which showed 69.5% identity to the Bacillus subtilis azoreductase YhdA. The enzyme was homotetrameric and molecular weight of each subunit was 20 kDa. It decolorized azo dyes with different structures. The Vmax for decolorization of congo red, methyl orange and methyl red was 14.7, 28.6, and 77.9 nmol/min/mg, respectively. The enzyme contained FMN as cofactor and used NADPH as the favored co-substrate. It was oxygen-insensitive, but the presence of reducing agents enhanced its activity, which is a new finding. The azoreductase expression in B. velezensis was found to be unaffected by addition of azo dyes, although azo dyes are known to induce azoreductase expression in few organisms. The enzyme was thermostable with melting temperature of 89.5°C and functioned in wide temperature range. Further, the enzyme was crystallized and its structure was solved. The structural basis of its functional attributes is discussed. In our knowledge, this is the first report on characterization of azoreductase enzyme from B. velezensis.

The FMN-dependent NADH-indigo reductase gene from the thermophilic bacterium Bacillus smithii was overexpressed in Escherichia coli. The expressed enzyme functioned as a highly thermostable indigo reductase that retained complete activity even after incubation at 100 °C for 10 min. Furthermore, B. smithii indigo reductase exhibited high stability over a wider pH range and longer storage periods compared with indigo reductases previously identified from other sources. The enzyme catalyzed the reduction of various azo compounds and indigo carmine. The crystal structures of the wild-type enzyme in complex with FMN/N-cyclohexyl-2-aminoethanesulfonate (CHES) and the Y151F mutant enzyme in complex with FMN were determined by the molecular replacement method and refined at resolutions of 1.97 and 1.95 A, respectively. Then, indigo carmine molecule was modeled into the active site using the molecular docking simulation and the binding mode of indigo carmine was elucidated. In addition, the structure of B. cohnii indigo reductase, which is relatively less stable than B. smithii indigo reductase, was constructed by homology modeling. The factor contributing to the considerably higher thermostability of B. smithii indigo reductase was analyzed by comparing its structure with that of B. cohnii indigo reductase, which revealed that intersubunit aromatic interactions (F105-F172′ and F172-F105′) may be responsible for the high thermostability of B. smithii indigo reductase. Notably, site-directed mutagenesis results showed that F105 plays a major role in the intersubunit aromatic interaction.

2,4,6-trinitrotoluene (TNT) is a persistent pollutant; for removing it from environment, the biodegradation becomes an economical and environmentally friendly alternative. However, TNT is difficult to naturally mineralize due to the stability of benzene ring. Interestingly, the hydride metabolite (Hˉ-TNT) of TNT can produce dearomatized products by the biotransformation of old yellow enzyme (OYE) family, which is highly different with TNT. It is a promising strategy for the ring-opening degradation of TNT. Here, we explore the biotransformation difference of TNT and Hˉ-TNT by OYE family. The results show that the electron-withdrawing ability of nitro groups makes them obtain the majority of negative charges of Hˉ-TNT, which promotes the strong interaction between the substrate and key residues. It in turn promotes the formation of π-π stacking configuration between Hˉ-TNT and the flavin mononucleotide (FMN) cofactor, which is a precondition of the aromatic ring reduction. As a result, the aromatic ring reduction occupies an absolute advantage, while the nitro reduction is suppressed. The conclusions are in line with previous understanding and can explain experimental phenomena. This work can provide a valuable reference for the combined strategy of mineralizing TNT.

We evaluated the protective effect of ademetionine, cytoflavin, and dihydroquercetin on activity of serum enzymes in rats treated with high doses of sodium valproate for 28 days. Ademetionine and cytoflavin produced the greatest protective effect, the effect of dihydroquercetin was less pronounced. In rats treated with ademetionine, AST activity decreased as soon as on day 7 and remained at this level until the end of the experiment; ALT, alkaline phosphatase, and γ-glutamyl transferase activities decreased on days 21 and 28 of the study. Cytoflavin produced similar effects, the effect of dihydroquercetin was observed on days 21 and 28 for AST, ALT, alkaline phosphatase and on day 28 for γ-glutamyl transferase. These results substantiate the use of hepatoprotective drugs in case of long-term treatment with anticonvulsants in patients with epilepsy.

In the past several decades, antibiotic drug resistance has emerged as a significant challenge in modern medicine due to the rise of many bacterial pathogenic strains resistant to all known antibiotics. At the same time, riboswitches have emerged as novel targets for antibacterial drug discovery. Here for the first time, we describe the design and applications of antisense oligonucleotides as antibacterial agents that target a riboswitch. The antisense oligonucleotides are covalently coupled with two different cell-penetrating peptides, penetrating Gram-positive and Gram-negative bacterial cells. We specifically target Flavin MonoNucleotide (FMN) riboswitches in

Methods for facile site-selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site-specific covalent attachment of a chromo- and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin-tag method resulted in efficient labeling of proteins in vitro , labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin-tag method that addresses previous limitations. We demonstrate that coexpression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin-labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable.