Vitamins | Riboflavin

FMN Phosphatase and FAD Pyrophosphatase in Rat Intestinal Brush Borders: Role in Intestinal Absorption of Dietary Riboflavin

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

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.

Investigation of electrochemical properties of FMN and FAD adsorbed on titanium electrode

AD= : Alzheimer disease; cAMP= : cyclic adenosine monophosphate; cGMP= : cyclic guanosine monophosphate; CREB= : cyclic adenosine monophosphate response element-binding protein; DRG= : dorsal root ganglion; Drp-1= : dynamin-related protein 1; EET= : epoxyeicosatrienoic acid; eNOS= : endothelial nitric oxide synthase; FAD= : flavin adenine dinucleotide; FMN= : flavin mononucleotide; GABA= : γ-aminobutyric acid; GADPH= : glyceraldehyde-3-phophate dehydrogenase; HETE= : hydroxyeicosatetraenoic acid; Hsp= : heat shock protein; iNOS= : inducible nitric oxide synthase; MELAS= : mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; MS= : multiple sclerosis; mtNOS= : mitochondrial variant of NOS; NADPH= : nicotinamide-adenine dinucleotide phosphate; NF-kB= : nuclear factor κB; NMDAR= : NMDA receptor; nNOS= : neuronal nitric oxide synthase; NO= : nitric oxide; NOS= : nitric oxide synthase; PD= : Parkinson disease; PKG= : protein kinase G; PNS= : peripheral nervous system; RNS= : reactive nitrogen species; SAH= : subarachnoid hemorrhage; SNO= : S-nitrosothiol; sGC= : soluble (cytosolic) guanylyl cyclase; VIP= : vasoactive intestinal polypeptide Nitric oxide (NO) is a gaseous chemical transmitter that is produced from the amino acid l-arginine by the members of the NO synthase (NOS) family of proteins. NO is involved in several important functions in the CNS and peripheral nervous system (PNS), including modulation of neurotransmission and regulation of local blood flow and immune responses. NO interacts with intracellular targets to trigger several signal transduction pathways. In the CNS, NO participates in synaptic modulation and plasticity, control of sleep, body temperature, and neurosecretion. In the PNS, NO mediates vasodilation and relaxation of the visceral smooth muscle. However, when produced in excess and in the setting of oxidative stress, NO becomes toxic, leading to formation of reactive nitrogen species (RNS), which cause cellular damage. These neurotoxic effects of NO derivatives may be involved in the pathogenesis of cerebral ischemia, inflammation, neoplasia, and neurodegenerative disorders. There are several comprehensive reviews on the physiologic and pathophysiologic roles of NO in the nervous system.1,–,12 ### Catalytic activity. The NOS family of enzymes catalyzes the oxidation of l-arginine to form l-citrulline and NO (figure 1). NOS is a dimeric enzyme; each monomer is composed of 2 distinct catalytic domains, an amino (NH2)-terminal oxygenase domain and a carboxy (COOH)-terminal reductase domain. The oxygenase domain is the binding site for heme, oxygen (O2), tetrahydrobiopterin (BH4), and l-arginine; the reductase domain binds flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and reduced nicotinamide-adenine dinucleotide phosphate (NADPH). The catalytic mechanism involves flavin-mediated electron transport from NADPH to the heme center, where O2 is reduced and incorporated into the guanidine nitrogen of l-arginine, producing NO and l-citrulline. Figure 1 General features of nitric oxide signaling Nitric oxide (NO*) is produced from arginine by action of NO synthase (NOS) in the presence of heme, oxygen (O2), tetrahydrobiopterin (BH4) as flavin adenine dinucleotide (FAD), flavin mononucleotide …

31] Luminometric determination of flavin adenine dinucleotide

We present a strategy for enhancing the intrinsic emission of the enzyme cofactors flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and nicotinamide adenine dinucleotide (NADH). Ensemble studies show that silver island films (SIFs) are the optimal metal enhanced fluorescence (MEF) substrates for flavins and gave emission enhancements of over 10-fold for both FAD and FMN. A reduction in the lifetime of FAD and FMN on SIFs was also observed. Thermally evaporated aluminum films on quartz slides were found to be the optimal MEF substrate for NADH and gave a 5-fold increase in the emission intensity of NADH. We present finite-difference time-domain (FDTD) calculations that compute the enhancement in the radiated power emitting from an excited state dipole emitting in the wavelength range of NADH in close proximity to an aluminum nanoparticle, and a dipole emitting in the emission wavelength of flavins next to a silver nanoparticle. These calculations confirm that aluminum serves as the optimal MEF substrate for NADH and silver was the optimal MEF substrate for flavins. This is because the plasmon resonance properties of aluminum lie in the UV-blue regime and that of silver lie in the visible region. We also present the results of single molecule studies on FMN which show SIFs can both significantly enhance the intrinsic emission from single FMN molecules, significantly reduce their lifetimes and also significantly reduce FMN blinking. This is the first report of the observation of MEF from cofactors both at the ensemble and single molecule level. We hope this study will serve as a platform to encourage the future use of metallic nanostructures to study cofactors using their intrinsic fluorescence to directly monitor enzyme binding reactions without the need of extrinsic labeling of the molecules.

Chapter 12 - Riboflavin

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.

Neutral red uptake by corneal epithelial cells and by injury-activated corneal stromal cells: II. Competitive inhibition by riboflavin derivatives

Effects of enhancing mitochondrial oxidative phosphorylation with reducing equivalents and ubiquinone on 1-methyl-4-phenylpyridinium toxicity and complex I–IV damage in neuroblastoma cells

Modulation of the subcellular levels of redox cofactors by Nudix hydrolases in chloroplasts

Characterization of Excited State Etheno-Fad: A Probe of the Role of Adenine in DNA Photolyase

Non-degradative extraction and simultaneous quantitation of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in foods by HPLC

The activity of cytochrome P450 enzymes decreases in older adults, which can lead to toxic effects from polypharmacy. Cytochromes P450 are the most significant enzymes involved in the metabolism of foreign compounds, including pharmaceutical substances. Vitamin B2, or riboflavin (RF), is a potent antioxidant that is vital for the body and participates in numerous enzyme-catalyzed redox reactions. RF is phosphorylated intracellularly to form flavin mononucleotide (FMN), which is further metabolized into flavin adenine dinucleotide (FAD). The active site of the NADPH-dependent cytochrome P450 reductase (CPR), a redox partner of CYP enzymes, is necessary for the catalytic functions of cytochromes P450. The active site of reductase is a complex formed by two types of vitamin B2, such as flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). In our study, we investigated the impact of the phosphorylated form of vitamin B2, FAD, and FMN on the catalytic activity of cytochrome P450 2C9 (CYP2C9) towards non-steroidal anti-inflammatory medications diclofenac and naproxen. It was shown that FAD significantly enhanced the catalytic efficiency of CYP2C9. The 4-hydroxylation of diclofenac was enhanced by 148 ± 10%. The O-demethylation of naproxen showed an increase of 120 ± 14%. Based on these data, we can assume that intake of vitamin B2 (riboflavin) improves catalytic efficiency of CYP2C9. This finding is essential for the modulation of catalytic activity of CYP2C9. The proposed electroanalytic approach is a sensitive and robust method for drug metabolism assay.