Presence Of Flavin Mononucleotide Research Articles

Metagenomic analysis of a bioreactor biofilm treating raw textile effluent and biochemical characterization of a novel azoreductase gene

Azo dyes are potent synthetic carcinogens that are widely used in the textile industries and their discharge into the surface water has detrimental consequences for human and ecosystem health. Oxidoreductases have been found to be involved in the metabolism of various xenobiotic compounds including azo dyes. Therefore, in this study shotgun metagenomic sequencing (SGMS) was used to bioprospect novel oxidoreductase genes in the biofilm of a fixed-film bioreactor treating raw textile effluent. According to the SGMS taxonomic and functional annotation, the bacteria found to be dominant in the bioreactor biofilm exhibited versatility and possessed genes that are capable of degrading azo dyes as well as other xenobiotics. A novel azoreductase gene (BM-AzoR) derived from the bioreactor biofilm metagenome was found to encode a previously uncharacterized FMN-dependent NADH azoreductase. In silico analysis of BM-AzoR gene encoded enzyme suggests that it is a thermostable and hydrophilic monomeric protein. The purified BM-AzoR is a flavin protein showing an optimal enzyme activity at pH 5 and 55 °C, respectively. The purified enzyme showed a specific azoreductase activity of 1.348 ± 0.12 U mg−1 in the presence of flavin mononucleotide (FMN) and preferred nicotinamide adenine dinucleotide (NADH) over nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. A molecular docking analysis validated the in vitro results by identifying Methyl Red as the best substrate for the BM-AzoR. The purified enzyme retained its activity in a variety of metal ions and solvents, indicating its potential application in azo dye bioremediation.

NADH Regeneration Promoted by Solar Light Using Gold Nanoparticles/Layered Double Hydroxides as Novel Photocatalytic Nanoplatforms

AbstractNicotinamide adenine dinucleotide NAD(P)H is a prolific cofactor in oxidoreductases biochemistry, but its high cost and the stoichiometric amounts motivates significant research for establishing efficient solutions for regenerating NAD(P)H from NAD(P)+. This study presents nanoparticles of gold/layered double hydroxides (AuNPs/LDH) as nanoplatforms with highly efficient photocatalytic response for solar‐light promoted NADH regeneration. The novel photocatalysts are obtained via self‐assembling of ZnAlLDH and AuNPs by using the manifestation of the “structural memory” of the anionic clay in gold salt solution. The chemical composition, structural, optical and nanotextural features of AuNPs/LDH have been assessed by transmission electron microscopy (TEM), energy dispersive X‐Ray Spectroscopy (EDX), powder X‐ray diffraction (XRD), and UV‐Vis Spectroscopy (UV‐Vis). It was found that, at pH 8 and in the presence of flavin mononucleotide (FMN) as a mediator and using water as an electron donor, the regeneration the NADH under solar light is defined by a rate of 20.5 μM/h. Furthermore, the regenerated NADH cofactor is tested in presence of Horseradish Peroxidase (HRP) enzyme to promote the hydrogen peroxide reduction.

Preparing Modified Hyaluronic Acid with Tunable Content of Vinyl Groups for Use in Fabrication of Scaffolds by Photoinduced Crosslinking

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.

Electrochemical Study on the Extracellular Electron Transfer Pathway from Shewanella Strain Hac319 to Electrodes.

Shewanella can transfer electrons to various extracellular electron acceptors. We electrochemically investigated the pathway of extracellular electron transfer from Shewanella strain Hac319 to electrodes. A resting cell suspension of Shewanella strain Hac319 containing lactate produced a steady-state sigmoidal wave in the presence of flavin mononucleotide (FMN) in cyclic voltammetry, but not in the absence of FMN. A harvested cell suspension without cell-washing also produced a similar catalytic wave without any external addition of free FMN. The midpoint potentials of the two sigmoidal waves were identical to the redox potential of free FMN. The data indicate that FMN secreted from the Shewanella strain Hac319 works as an electron-transfer mediator from the cell to electrodes. An addition of cyanide to a resting cell suspension of Shewanella strain Hac319 increased the rate of the FMN reduction in the presence of lactate, while it decreased the respiration rate. By considering the fact that cyanide is coordinated to the heme moiety of hemoproteins and shifts the redox potential to the negative potential side, the data indicate that the electron derived from lactate is predominantly transferred in a down-hill mode from an electron donor with a redox potential more negative than that of FMN without going through outer membrane cytochromes c molecules.

Electrochemical Synthesis and Characterization of Flavin Mononucleotide‐Exfoliated Pristine Graphene/Polypyrrole Composites

AbstractThe electrochemical generation of graphene‐polypyrrole composite films is presented here by using pristine, oxide‐free graphene flakes obtained through the direct exfoliation of graphite in water with flavin mononucleotide (FMN) as a colloidal stabilizer (FMN−G‐PPy films): an environmentally friendly methodology. The best potential and current ranges for the electropolymerization of pyrrole in the presence of FMN−G, trying to avoid parallel over‐oxidation/degradation processes, were determined from voltammetric responses of the different solutions. Then, a parallel study of the electrogeneration of FMN−G‐PPy films on clean Pt electrodes by using cyclic voltammetry, potentiostatic or galvanostatic conditions, was performed. The subsequent electrochemical characterization of the resulting films and parallel energy‐dispersive X‐ray spectroscopy analysis of different oxidized states revealed the separate oxidation/reduction of graphene and polypyrrole inside the material. Graphene reactions (n‐doping) drive the exchange of cations, whereas polypyrrole reactions (p‐doping) drive the exchange of anions. The specific charge storage in FMN−G‐PPy, 207 mC mg−1, is higher than that stored in polypyrrole, 141 mC mg−1, indicating some synergy. An unexpected high content of anions was found in deeply reduced materials. The surface morphology of the oxidized and reduced materials generated by different methodologies was studied by using scanning electron microscopy.

440 - Electron Transfer Activity of Mitochondrial Outer Membrane Protein MitoNEET

Mitochondrial outer membrane protein MitoNEET, a primary target of type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. The protein exists as a homodimer with each monomer containing a redox active [2Fe-2S] cluster. Here we report that mitoNEET has a specific interaction with flavin mononucleotide (FMN). In the presence of flavin reductase, FMNH2 reduced by NADH can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic conditions. Under aerobic conditions, however, the reduced mitoNEET [2Fe-2S] clusters are constantly re-oxidized by oxygen with concomitant oxidation of NADH in the presence of FMN and flavin reductase. The reduced mitoNEET [2Fe-2S] clusters can also reduce ubiquinone-2, a ubiquinone-10 analog, under aerobic or anaerobic conditions, indicating that mitoNEET has the ability to transfer electron from NADH to the membrane-bound ubiquinone in mitochondria. The [2Fe-2S] cluster is required for the electron transfer activity of mitoNEET, as apo-form mitoNEET has no activity to mediate oxidation of NADH or reduction of ubiquinone. The pioglitazone analog NL-1, which has a specific binding affinity for mitoNEET, effectively inhibits the electron transfer activity of the mitoNEET [2Fe-2S] clusters. The results suggest that mitoNEET is a novel electron transfer protein that may directly regulate energy metabolism in mitochondria.

Optimization of 3', 4'-Anhydrovinblastine Synthesis in vitro Using Crude Extracts of Catharanthus roseus Irradiated with Near-Ultraviolet Light

Dimeric indole alkaloids (DIAs), such as vinblastine and vincristine, found in Catharanthus roseus are used clinically as antitumor drugs. A stable supply of DIAs is desired because these alkaloids are very expensive due to their low abundance in plants. A coupling reaction between catharanthine (CAT) and vindoline (VID) is the rate-limiting step of DIAs biosynthesis in planta. 3', 4'-Anhydrovinblastine (AVLB), the product of the coupling reaction, is the precursor of CAT and VID. Therefore, an effective AVLB production system is greatly required. Previously we found that the coupling reaction of CAT and VID to produce AVLB occurred in the presence of flavin mononucleotide and manganese ion (II) by irradiation with near-ultraviolet light at a peak of 370 nm without the presence of any enzyme. In this study, we investigated the effects of organic solvents on this non-enzymatic reaction. We show that the addition of 10% methanol to the reaction mixture permitted the preparation of a highly concentrated substrate solution, resulting in a high yield of AVLB by the coupling reaction. Conditions for the coupling reaction in 10% methanol solution were optimized. We also confirmed that the coupling reaction could occur in crude extracts of C. roseus obtained by organic solvent extraction. These findings suggest a method to produce DIAs on a large scale with reduced production costs.

The reduced flavin-dependent monooxygenase SfnG converts dimethylsulfone to methanesulfinate

The biochemical pathway through which sulfur may be assimilated from dimethylsulfide (DMS) is proposed to proceed via oxidation of DMS to dimethylsulfoxide (DMSO) and subsequent conversion of DMSO to dimethylsulfone (DMSO2). Analogous chemical oxidation processes involving biogenic DMS in the atmosphere result in the deposition of DMSO2 into the terrestrial environment. Elucidating the enzymatic pathways that involve DMSO2 contribute to our understanding of the global sulfur cycle. Dimethylsulfone monooxygenase SfnG and flavin mononucleotide (FMN) reductase MsuE from the genome of the aerobic soil bacterium Pseudomonas fluorescens Pf0-1 were produced in Escherichia coli, purified, and biochemically characterized. The enzyme MsuE functions as a reduced nicotinamide adenine dinucleotide (NADH)-dependent FMN reductase with apparent steady state kinetic parameters of Km = 69 μM and kcat/Km = 9 min−1 μM −1 using NADH as the variable substrate, and Km = 8 μM and kcat/Km = 105 min−1 μM −1 using FMN as the variable substrate. The enzyme SfnG functions as a flavoprotein monooxygenase and converts DMSO2 to methanesulfinate in the presence of FMN, NADH, and MsuE, as evidenced by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The results suggest that methanesulfinate is a biochemical intermediate in sulfur assimilation.

Redox Linked Flavin Sites in Extracellular Decaheme Proteins Involved in Microbe-Mineral Electron Transfer.

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX8C disulfide that, when substituted for AX8A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation of a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.

Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion

Here, we determined the electron spin resonance (ESR) spectra of standard reaction mixtures (I) containing 25 μM flavin mononucleotide (FMN), 0.018% tea tree (Melaleuca alternifolia) oil, 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and 1.0 mM FeSO4(NH4)2SO4 irradiated with 436 nm visible light (7.8 J/cm2). Prominent ESR signals (αN = 1.58 mT and αHβ = 0.26 mT) were detected, suggesting that free radicals form in the standard reaction. In order to know whether singlet oxygen (1O2) is involved in the radical formation or not, ESR measurement was performed for the standard D2O reaction mixture (I) which contained 25 μM FMN, 0.0036% tea tree oil, 1.9 M acetonitrile-d3, 20 mM phosphate buffer (pH 7.4), 0.1 M 4-POBN and 1.0 mM FeSO4 in D2O. The ESR peak height of the standard D2O reaction increased to 169 ± 24% of the control. Thus, 1O2 seems to be involved in the formation of the radicals because D2O increases the lifetime of singlet oxygen. High-performance liquid chromatography-ESR-mass spectrometry analyses detected 1-methylethyl and methyl radicals in the standard reaction. The radicals appear to form through the reaction of ferrous ion with α-terpinene endoperoxide (ascaridole), which generated from the reaction of α-terpinene with 1O2. The 1-methylethyl and methyl radicals may exert a pro-oxidant effect under these conditions.

Molecular sensing by the aptamer domain of the FMN riboswitch: a general model for ligand binding by conformational selection

Understanding the nature of the free state of riboswitch aptamers is important for illuminating common themes in gene regulation by riboswitches. Prior evidence indicated the flavin mononucleotide (FMN)-binding riboswitch aptamer adopted a ‘bound-like’ structure in absence of FMN, suggesting only local conformational changes upon ligand binding. In the scope of pinpointing the general nature of such changes at the nucleotide level, we performed SHAPE mapping experiments using the aptamer domain of two phylogenetic variants, both in absence and in presence of FMN. We also solved the crystal structures of one of these domains both free (3.3 Å resolution) and bound to FMN (2.95 Å resolution). Our comparative study reveals that structural rearrangements occurring upon binding are restricted to a few of the joining regions that form the binding pocket in both RNAs. This type of binding event with minimal structural perturbations is reminiscent of binding events by conformational selection encountered in other riboswitches and various RNAs.

Synthesis of Anti-Tumor Dimeric Indole Alkaloids inCatharanthus roseusWas Promoted by Irradiation with Near-Ultraviolet Light at Low Temperature

We have found that coupling between catharanthine and vindoline occurs non-enzymatically in the presence of flavin mononucleotide and manganese ions with near-ultraviolet light irradiation in vitro. The present study found that the concentrations of catharanthine and vindoline in Catharanthus roseus decreased and those of dimeric indole alkaloids increased under near-ultraviolet light at 4 degrees C. It indicates that this coupling reaction at 4 degrees C occurs non-enzymatically.

Flavin mononucleotide mediated electron pathway for microbial U(vi) reduction

Microbial reduction of U(VI) is an important phenomenon affecting uranium mobility in the subsurface environments. Elucidation of its mechanism is necessary for predicting uranium migration and for applying environmental remediation. In this study, we have examined the electron pathway for the U(VI) reduction mediated by flavin mononucleotide (FMN), which is secreted by Shewanella species. The cyclic voltammetry (CV) and photo-electrochemical methods with an optically transparent thin-layer electrode (OTTLE) cell were utilized in investigating in vitro the electron transfer reactions that take place between FMN and U(VI). The CV measurements of U(VI) were carried out in a citrate and Tris-HCl buffer both with and without FMN. A scarce U(VI) reduction current was observed in the absence of the FMN. To the contrary, a catalytic U(VI) reduction current was observed in the presence of FMN at the redox potential of the FMN. The reduction current increased with an increase in the concentration of the U(VI). The reduced form of the U was confirmed to be U(VI) by the photo-electrochemical analysis using the OTTLE cell. The results demonstrated that FMN acts as a mediator in the electro-reduction of U(VI) to U(iv). In addition, in vivo bio-reduction experiments on U(VI) with Shewanella putrefaciens revealed that the addition of FMN accelerated the reduction rate of U(VI). These results indicate that the bio-reduction of U(VI) by the Shewanella species can be catalyzed by FMN secreted from the cells.

1H NMR study of the hetero-association of flavin-mononucleotide with mutagenic dyes: ethidium bromide and proflavine

The hetero-association of the vitamin B2 derivative, flavin-mononucleotide (FMN), with a mutagenic dye, ethidium bromide (EB) or proflavine (PF), has been studied by 1D and 2D 500 MHz 1H NMR spectroscopy. The variations of proton chemical shifts of both the vitamin and dye as a function of concentration and temperature were analysed in terms of the structural and thermodynamical properties of the FMN–EB and FMN–PF complexes in solution. The structures of the complexes were also investigated by observed intermolecular ROE contacts and molecular mechanics calculations. The results show that the 1 : 1 hetero-association complexes in solution are more stable than the self-association complexes, which is consistent with formation of an intermolecular hydrogen-bond in the hetero-complexes of FMN–EB and FMN–PF. Hence it is possible that the toxicity of aromatic molecules such as EB and PF may be reduced in vitro by the presence of FMN, partly because of the known antimutagenic action of FMN and partly because it has been shown in this work that there is an effective intermolecular association between the mutagens and the vitamin.

Staphylococcus aureus NfrA (SA0367) Is a Flavin Mononucleotide-Dependent NADPH Oxidase Involved in Oxidative Stress Response

The NfrA protein, a putative essential oxidoreductase in the soil bacterium Bacillus subtilis, is induced under heat shock and oxidative stress conditions. In order to characterize the function of an homologous NfrA protein in Staphylococcus aureus, an nfrA deletion strain was constructed, the protein was purified, the enzymatic activity was determined, and the transcriptional regulation was investigated. The experiments revealed that NfrA is not essential in S. aureus. The purified protein oxidized NADPH but not NADH, producing NADP in the presence of flavin mononucleotide, suggesting that NfrA is an NADPH oxidase in S. aureus. In addition, the NfrA enzyme showed nitroreductase activity and weak disulfide reductase activity. Transcription was strongly induced by ethanol, diamide, and nitrofurantoin. Hydrogen peroxide induced nfrA transcription only at high concentrations. The expression of nfrA was independent of the alternative sigma factor sigma(B). Furthermore, the transcriptional start site was determined, which allowed identification of a PerR box homologous sequence upstream of the nfrA promoter. The observations presented here suggest that NfrA is a nonessential NADPH oxidoreductase which may play a role in the oxidative stress response of S. aureus, especially in keeping thiol-disulfide stress in balance.

The Structure of Chorismate Synthase Reveals a Novel Flavin Binding Site Fundamental to a Unique Chemical Reaction

The crystal structure of chorismate synthase (CS) from Streptococcus pneumoniae has been solved to 2.0 Å resolution in the presence of flavin mononucleotide (FMN) and the substrate 5-enolpyruvyl-3-shikimate phosphate (EPSP). CS catalyses the final step of the shikimate pathway and is a potential therapeutic target for the rational design of novel antibacterials, antifungals, antiprotozoals, and herbicides. CS is a tetramer with the monomer possessing a novel β-α-β fold. The interactions between the enzyme, cofactor, and substrate reveal the structural reasons underlying the unique catalytic mechanism and identify the amino acids involved. This structure provides the essential initial information necessary for the generation of novel anti-infective compounds by a structure-guided medicinal chemistry approach.

MioC Is an FMN-binding Protein That Is Essential forEscherichia coli Biotin Synthase Activity in Vitro

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.

Identification and expression of a cDNA for human glycolate oxidase

The isolation and expression of a human liver cDNA encoding a 43 kDa protein with glycolate oxidase activity is described. The cDNA (HAO1) is 1128 bp and has a 1113 bp open reading frame. Northern blot analysis detects a 1.8 kb mRNA with expression restricted to the liver. The 370 amino acid protein has 89% sequence similarity to glycolate oxidase from mouse liver and 53% similarity to the spinach and Arabidopsis enzymes. The protein has glycolate oxidase activity in vitro, as measured by the reduction of 2,6-dichlorophenolindophenol in the presence of flavin mononucleotide and glycolate. The genomic sequence is contained within eight exons and encompasses approximately 57 kb of chromosome 20p12. This paper offers the first full functional description of a gene encoding human glycolate oxidase.

Design of allosteric hammerhead ribozymes activated by ligand-induced structure stabilization.

Ribozymes can function as allosteric enzymes that undergo a conformational change upon ligand binding to a site other than the active site. Although allosteric ribozymes are not known to exist in nature, nucleic acids appear to be well suited to display such advanced forms of kinetic control. Current research explores the mechanisms of allosteric ribozymes as well as the strategies and methods that can be used to create new controllable enzymes. In this study, we exploit the modular nature of certain functional RNAs to engineer allosteric ribozymes that are activated by flavin mononucleotide (FMN) or theophylline. By joining an FMN- or theophylline-binding domain to a hammerhead ribozyme by different stem II elements, we have identified a minimal connective bridge comprised of a G.U wobble pair that is responsive to ligand binding. Binding of FMN or theophylline to its allosteric site induces a conformational change in the RNA that stabilizes the wobble pair and ultimately favors the active form of the catalytic core. These ligand-sensitive ribozymes exhibit rate enhancements of more than 100-fold in the presence of FMN and of approximately 40-fold in the presence of theophylline. An adaptive strategy for modular rational design has proven to be an effective approach to the engineering of novel allosteric ribozymes. This strategy was used to create allosteric ribozymes that function by a mechanism involving ligand-induced structure stabilization. Conceivably, similar engineering strategies and allosteric mechanisms could be used to create a variety of novel allosteric ribozymes that function with other effector molecules.

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.