flavin-adenine-dinucleotide and 4-6-dinitro-o-cresol

Natural products are usually highly complicated organic molecules with special scaffolds, and they are an important resource in medicine. Natural products with complicated structures are produced by enzymes, and this is still a challenging research field, its mechanisms requiring detailed methods for elucidation. Flavin adenine dinucleotide (FAD)-dependent monooxygenases (FMOs) catalyze many oxidation reactions with chemo-, regio-, and stereo-selectivity, and they are involved in the synthesis of many natural products. In this review, we introduce the mechanisms for different FMOs, with the classical FAD (C4a)-hydroperoxide as the major oxidant. We also summarize the difference between FMOs and cytochrome P450 (CYP450) monooxygenases emphasizing the advantages of FMOs and their specificity for substrates. Finally, we present examples of FMO-catalyzed synthesis of natural products. Based on these explanations, this review will expand our knowledge of FMOs as powerful enzymes, as well as implementation of the FMOs as effective tools for biosynthesis.

Topics: Biological Products; Cytochrome P-450 Enzyme System; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Oxygenases

Flavins are blue-light-absorbing chromophores with rich redox activity. Biologically, the most important are riboflavin (vitamin B

Topics: Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Organic Chemicals; Oxidation-Reduction; Riboflavin

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (

Topics: Electron Transport; Electron-Transferring Flavoproteins; Flavin-Adenine Dinucleotide; Flavins; Flavoproteins; Oxidation-Reduction

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.

Topics: Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Flavoproteins; Oxidation-Reduction

The flavin adenine dinucleotide (FAD) is an indispensable coenzyme in live cells. It acts as a catalyst in many redox responsive metabolic reactions, including oxidative phosphorylation in mitochondria. The real-time monitoring of flavin is important to understand the disorder in the metabolic process, redox system, etc. Thus, we have developed a fluorescent probe

Topics: Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Topics: Anions; Catalytic Domain; Dinitrocresols; Electron Transport; Flavin-Adenine Dinucleotide; Flavins; Flavoproteins; Kinetics; Light; Models, Molecular; Molecular Dynamics Simulation; Oxidation-Reduction; Oxidoreductases; Spectrophotometry

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade

Topics: Adenine; Density Functional Theory; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Spectrometry, Fluorescence

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.

Topics: Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Organic Chemicals; Riboflavin; Solvents

Xanthone-containing natural products display diverse pharmacological properties. The biosynthetic mechanisms of the xanthone formation have not been well documented. Here we show that the flavoprotein monooxygenase FlsO1 in the biosynthesis of fluostatins not only functionally compensates for the monooxygenase FlsO2 in converting prejadomycin to dehydrorabelomycin, but also unexpectedly converts prejadomycin to xanthone-containing products by catalyzing three successive oxidations including hydroxylation, epoxidation and Baeyer-Villiger oxidation. We also provide biochemical evidence to support the physiological role of FlsO1 as the benzo[b]-fluorene C5-hydrolase by using nenestatin C as a substrate mimic. Finally, we resolve the crystal structure of FlsO1 in complex with the cofactor flavin adenine dinucleotide close to the "in" conformation to enable the construction of reactive substrate-docking models to understand the basis of a single enzyme-catalyzed multiple oxidations. This study highlights a mechanistic perspective for the enzymatic xanthone formation in actinomycetes and sets an example for the versatile functions of flavoproteins.

Topics: Catalysis; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavoproteins; Isoquinolines; Mixed Function Oxygenases; Naphthoquinones; Organic Chemicals; Xanthones

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Topics: Bacterial Proteins; Biocatalysis; Cadaverine; Catalytic Domain; Deferoxamine; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Holoenzymes; Hydroxylation; Kinetics; Mixed Function Oxygenases; NADP; Ornithine; Oxidation-Reduction; Siderophores; Streptomyces

Cryptochromes are ubiquitous flavin-binding light sensors closely related to DNA-repairing photolyases. The animal-like cryptochrome

Topics: Chlamydomonas; Chlamydomonas reinhardtii; Color; Cryptochromes; Deoxyribodipyrimidine Photo-Lyase; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Light; Riboflavin; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

It has been shown that spontaneous release of non-covalent flavins (from flavoenzymes) begins after isolation of mitochondria from rat liver, which is hydrolyzed to riboflavin. This process is stopped by 1 mM EDTA in the incubation medium. In the presence of NADH, deflavinization of flavoproteins leads to formation of superoxide by at least of three processes. The first of these occurs in complex I as a result of the spontaneous release of FMN from the active center. This process is inhibited by adenosine and guanosine phosphates, as well as NAD, but amplified by nicotinamide. The second process is associated with enzymatic hydrolysis of FAD and FMN to riboflavin; it is blocked by EDTA, AMP, NA, NAD. The third process is associated with non-enzymatic hydrolysis of FAD by iron ions in matrix; it is blocked by EDTA and AMP.

Topics: Adenine Nucleotides; Animals; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Guanine Nucleotides; Homeostasis; Hydrolysis; Ions; Iron; Luminescence; Mitochondria, Liver; Niacinamide; Rats; Rats, Wistar; Riboflavin; Superoxides

The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.

Topics: Adenosine Diphosphate Ribose; Cations; Cytosol; Dinitrocresols; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Mitochondria; NAD; Potassium; Pyrophosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

Involvement of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding retinal pigment epithelium (RPE). Optimizations led to detection efficiency of 0.1 pmol for FAD and FMN while 0.01 pmol for riboflavin. Interestingly, levels of FAD and FMN in the RPE were found to be 1.7- and 12.5-fold higher than their levels in the retina, respectively. Both FAD and FMN levels in the RPE and retina gradually decline with age and preceded the age-dependent drop in the functional competence of the retina as measured by electroretinography. Further, quantifications of retinal levels of FAD and FMN in different mouse models of retinal degeneration revealed differential metabolic requirements of these two factors in relation to the rate and degree of photoreceptor degeneration. We also found twofold reductions in retinal levels of FAD and FMN in two mouse models of diabetic retinopathy. Altogether, our results suggest that retinal levels of FAD and FMN can be used as potential markers to determine state of health of the retina in general and more specifically the photoreceptors.

Topics: Aging; Animals; Chromatography, High Pressure Liquid; Dinitrocresols; Fasting; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Homeostasis; Light; Mice, Inbred C57BL; Retina; Retinal Degeneration; Retinal Pigment Epithelium

Thermal dependent conformational changes of xanthine oxidase (XOD) were studied using sensitive and non-destructive methods like fluorescence spectroscopy and molecular modeling in the temperature range of 25-85°C. Intrinsic fluorescence studies showed that the microenvironment of tryptophan and tyrosine residues becomes more exposed to solvent as the temperature increased up to 85°C, whereas in case of flavin cofactor is rather conserved. At higher temperatures, the flavin adenine dinucleotide is displaced from the core of the protein, but is not fully released as shown by the Stern Volmer quenching constant and accessible fraction of the cofactor. Anyway, no significant changes in the structure of XOD monomer were identified after running molecular dynamics simulations at temperatures 25°C, 65°C and 85°C. Therefore, we can conclude that the most important changes in the protein structure at thermal treatment mainly consist on molecular aggregation and dissociation events.

Topics: Animals; Cattle; Circular Dichroism; Dinitrocresols; Flavin-Adenine Dinucleotide; Hot Temperature; Milk; Molecular Dynamics Simulation; Protein Conformation; Protein Multimerization; Protein Subunits; Spectrometry, Fluorescence; Tryptophan; Tyrosine; Xanthine Oxidase

Topics: Arabidopsis; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Hydrolases; Riboflavin; Uracil Nucleotides

Methionine synthase reductase (MSR) and cytochrome P450 reductase (CPR) transfer reducing equivalents from NADPH via an FAD and FMN cofactor to a redox partner protein. In both enzymes, hydride transfer from NADPH to FAD requires displacement of a conserved tryptophan that lies coplanar to the FAD isoalloxazine ring. Swapping the tryptophan for a smaller aromatic side chain revealed a distinct role for the residue in regulating MSR and CPR catalysis. MSR W697F and W697Y showed enhanced catalysis, noted by increases in kcat and k(cat)/K(m)(NADPH) for steady-state cytochrome c(3+) reduction and a 10-fold increase in the rate constant (k(obs1)) associated with hydride transfer. Elevated primary kinetic isotope effects on k(obs1) for W697F and W697Y suggest that preceding isotopically insensitive steps like displacement of W697 are less rate determining. MSR W697Y, but not MSR W697F, showed detectable formation of the disemiquinone intermediate, indicating that the polarity of the aromatic side chain influences the rate of interflavin electron transfer. By contrast, the CPR variants (W676F and W676Y) displayed modest decreases in cytochrome c(3+) reduction, a 30- and 3.5-fold decrease in the rate of FAD reduction, accumulation of a FADH2 -NADP(+) charge-transfer complex and dramatically suppressed rates of interflavin electron transfer. We conclude for MSR that hydride transfer is 'gated' by the free energy required to disrupt dispersion forces between the FAD isoalloxazine ring and W697. By contrast, the bulky indole ring of W676 accelerates catalysis in CPR by lowering the energy barrier for displacement of the oxidized nicotinamide ring coplanar with the FAD.

Topics: Amino Acid Substitution; Amino Acids, Aromatic; Catalysis; Cytochrome c Group; Dinitrocresols; Electron Transport; Escherichia coli; Ferredoxin-NADP Reductase; Flavin-Adenine Dinucleotide; Kinetics; Mutagenesis, Site-Directed; NADP; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Plasmids; Protein Structure, Tertiary; Recombinant Proteins; Thermodynamics; Titrimetry; Tryptophan

Topics: Animals; Cattle; Chemical Precipitation; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Fluorometry; Muscles; Myocardium; Photometry; Research; Riboflavin; Spectrophotometry; Swine

Topics: Amino Acid Oxidoreductases; Amino Acids; D-Amino-Acid Oxidase; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Organic Chemicals; Research; Spectrophotometry

Topics: Animals; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Glutathione Reductase; Oxidoreductases; Rats

Topics: Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Folic Acid; Humans; Liver; Oxidoreductases; Riboflavin; Tetrahydrofolates

Topics: Adenine; Dinitrocresols; Flavin-Adenine Dinucleotide; Flavins; Laboratories

Topics: Animals; Biochemical Phenomena; Biochemistry; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Myocardium; Organic Chemicals; Research; Swine

Topics: Dinitrocresols; Electron Transport; Electron Transport Complex IV; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Metabolism; Mitochondria; NAD; NADH Dehydrogenase; Oxidoreductases; Peptide Hydrolases; Research; Riboflavin; Spectrophotometry

Topics: Barbiturates; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; NAD; NADP; Oxidation-Reduction; Pharmacology; Phenobarbital; Ultraviolet Rays

Topics: Coenzymes; Dinitrocresols; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Riboflavin

Topics: Biochemical Phenomena; Coenzymes; Dinitrocresols; Escherichia coli; Flavin-Adenine Dinucleotide; Flavins; Organic Chemicals

Topics: Coenzymes; Dinitrocresols; Electroencephalography; Flavin-Adenine Dinucleotide; Flavins; Humans; Organic Chemicals; Riboflavin

Topics: Acetylcholine; Adenine; Choline; Dinitrocresols; Flavin-Adenine Dinucleotide; Nucleotides