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At least 649 records · Page 36Linked to original sources
PubMed1983

Enzymatic synthesis of flavin adenine dinucleotide.

A new and simple enzymatic method for the synthesis of flavin adenine dinucleotide (FAD) from flavin mononucleotide by the transadenylylation reaction using microbial cells is described. Among various microorganisms tested, Artherobacter globiformis IFO 12138 and two soil bacteria were selected as useful enzyme sources. Under suitable reaction conditions, the amount of FAD synthesized was 2.25 mumol/mL with cells of A. globiformis. The transadenylylation reaction could be coupled with the ATP supplying system through a glycolysis process with yeast.

Arthrobacter↗
PubMed1986

The EH2 reduced intermediate of glutathione reductase contains oxidised flavin-while EH4 does not.

Glutathione reductase is a flavoprotein whose x-ray structure has been established. Functional data and the x-ray structure are consistent with a mechanism of reaction in which NADPH reacts with the enzyme to produce a two electron, EH2, and four electron, EH4, intermediate. The former is competent for the transfer of electrons to the substrate glutathione. Several structures are possible for the two NADPH intermediates; in order to aid in the determination of the structure of these intermediates, we have determined their resonance Raman spectra at two excitation frequencies. These studies establish that the EH2 intermediate is an oxidized flavin species while the EH4 species is not. Furthermore, the most likely structure for EH2 involves a charge transfer donation of electrons from the anion of cys-63 to the N5 position of flavin.

Flavins↗
PubMed1982

Cardiac sensitivity to the inhibitory effects of chlorpromazine, imipramine and amitriptyline upon formation of flavins.

Chlorpromazine, imipramine and amitriptyline, drugs structurally related to riboflavin, each inhibited the formation in vivo of flavin adenine dinucleotide (FAD) from riboflavin in rat heart at 2-5 mg/kg body weight, doses comparable on a weight basis to those used clinically. All three drugs inhibited FAD formation in heart within 5 hr after a single dose of 25 mg/kg. Chlorpromazine under these conditions also inhibited FAD formation in liver, cerebrum and cerebellum. A series of psychoactive agents structurally unrelated to riboflavin did not inhibit flavin formation in the organs tested. These findings indicate that the inhibitory effects of the drugs studied have organ specificity with respect to FAD formation.

Amitriptyline↗
PubMed1990

Flavin analogs with antimalarial activity as glutathione reductase inhibitors.

10-(4'-Chlorophenyl)-3-methylflavin has antimalarial activity in vitro and in vivo (Cowden et al., J Med Chem 31: 799, 1988). This flavin analog and two of its derivatives were found to inhibit the antioxidant flavoenzyme glutathione reductase from human erythrocytes in its isolated form as well as in hemolysates. The mixed-type inhibition was completely reversible, the Ki-values being of the order of 1 microM. Surprisingly, the drugs were not competitive with FAD, but with GSSG, one of the enzyme's substrates. Malaria parasite glutathione reductase, extracted from Plasmodium falciparum, could also be inhibited by the compounds. Studies on the effects of the substances on P. falciparum in vitro, which were demonstrated morphologically and by growth inhibition, confirmed previous observations with 10-(4'-chlorophenyl)-3-methylflavin and showed similar parasiticidal characteristics for the two new derivatives. The activities of five other erythrocytic enzymes tested were not impaired by the drugs, nor was the nucleotide metabolism of erythrocytes and/or parasites significantly changed. Permeation into red blood cells was demonstrated for one compound by 19F-NMR-spectroscopy. Inhibition of glutathione reductase might contribute to, or account for, the antimalarial activity of this group of flavin analogs.

Animals↗
PubMed1992

Use of thiocarbamides as selective substrate probes for isoforms of flavin-containing monooxygenases.

The oxidation of thiourea, phenylthiourea, 1,3-diphenylthiourea, 1,3-bis-(3,4-dichlorophenyl)-2-thiourea and 1,1-dibenzyl-3-phenyl-2-thiourea was measured in reactions catalyzed by purified pig liver flavin-containing monooxygenase (FMO-1) and by microsomal fractions isolated from pig, guinea pig, chicken, rat and rabbit tissues. The reactions, followed by measuring substrate-dependent thiocholine oxidation [Guo and Ziegler, Anal Biochem 198: 143-148, 1991], were carried out in the presence of 2 mM 1-benzylimidazole to minimize potential interference from reactions other than those catalyzed by isoforms of the flavin-containing monooxygenase (FMO). While at saturating substrate concentrations the Vmax for purified FMO-1 catalyzed oxidation of all five thiocarbamides was essentially constant, velocities for the microsomal catalyzed reactions varied not only with tissue and species but also with the van der Waals' surface area of the thiocarbamide. Rat liver, rat kidney and rabbit liver microsomes failed to catalyze detectable oxidation of thiocarbamides larger than 1,3-diphenylthiourea and lung microsomes from a female rabbit only accepted substrates smaller than 1,3-diphenylthiourea. On the other hand, liver microsomes from chickens, pigs and guinea pigs catalyzed the oxidation of larger thiocarbamides, but the rates decreased with increasing substrate size and chicken liver microsomes showed no detectable activity with the largest thiocarbamide tested. To define more precisely the parameters affecting thiocarbamide substrate specificity of microsomal preparations, activities present in detergent extracts of guinea pig liver microsomes were separated into three distinct fractions. The substrate specificities of these partially purified fractions were different and consistent with the difference observed with microsomal catalyzed reactions. This strongly suggests that thiocarbamides that differ in size may be useful probes for measuring the number of activities of FMO isoforms in crude tissue preparations.

Animals↗
PubMed1990

Structural studies on three flavin-interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria.

The flavoprotein (Fp) subunit of mitochondrial complex II contains covalently bound FAD as a prosthetic group. In this study, the primary structure of the flavin-bound tryptic peptide from the Fp subunit of Ascaris complex II was determined and found to be highly similar to those of the corresponding flavin-binding regions of bovine heart and bacterial Fp subunits. Furthermore, the Ascaris Fp subunit was shown to contain two regions exhibiting striking sequence similarity to the segments that have been predicted to interact noncovalently with the AMP moiety of FAD in bacterial Fp subunits. The conservation of these two regions also in the mitochondrial Fp subunit suggests their functional importance.

Amino Acid Sequence↗
PubMed1979

The measurement of subnanosecond fluorescence decay of flavins using time-correlated photon counting and a mode-locked Ar ion laser.

A system is described consisting of a mode-locked Ar ion laser and time-resolved photon-counting electronics. The system is capable of measuring fluorescence lifetimes in the subnanosecond time domain. The Ar ion laser is suitable for the excitation of flavins, since the available laser wavelengths encompass the first absorption band of the yellow chromophore. Due to the high radiation density and the short pulse, both the time and wavelength resolution of the fluorescence of very weakly emitting compounds can be measured. Experiments have been described for flavin models exhibiting single and multiple modes of decay. In these examples lifetimes were determined both from deconvolved decay curves and from direct analysis of the tail of the curve, where no interference of the exciting pulse is encountered. Both determinations showed very good agreement. Due to the highly polarized laser light the decay of the emission anisotropy could be measured directly after the exciting pulse. In principle, fast rotational motions might be detected. An anisotropy measurement conducted with a flavoprotein with a noncovalently attached FAD is presented.

Erythrosine↗
PubMed1984

Modification of redox equilibria between heme and flavin within yeast flavocytochrome b2 (L-lactate cytochrome c reductase) upon binding of pyruvate, the reaction product.

Direct determinations of the concentration of semiquinone spin in redox equilibrium with the cytochrome b2 moiety were carried out at room temperature, in the presence of added pyruvate or in its absence. Results show that redox potentials of the one-electron couples of the prosthetic flavin are markedly affected by binding of pyruvate, the reaction product in the oxidation of L-lactate. The proportion of flavin semiquinone nearly reaches then 100 per cent.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Flavin adenine dinucleotide is a major endogenous fluorophore in the inner ear.

When illuminated with visible light, hair cells can exhibit autofluorescence (Lewis et al. [1982] Science 215, 1641-1643) concentrated in the basal pole near the synapses (Sento and Furukawa [1987] J. Comp. Neurol. 258, 352-367). The autofluorescence is enhanced by formaldehyde. The level of fluorescence is high enough to interfere with fluorescence microscopy of hair cells and to suggest that the fluorescent substance might have a particular role in hair-cell function. To identify this substance, we extracted a substance with formaldehyde-enhanced fluorescence from the inner ears of goldfish and purified it chromatographically. The substance copurified with FAD and had the same fluorescence emission spectrum. Two further results supported the identity of the endogenous fluorescent substance with FAD. First, as is the case with flavins, the autofluorescence in inner ear tissue examined within a few hours after fixation was reduced by addition of dithionite. Second, as is the case with the formaldehyde-enhanced fluorophore, the fluorescence of FAD was enhanced by formaldehyde. FAD accounted for 90% of flavins in goldfish inner ears; its concentration in the sensory epithelium was estimated to be about 30 nmol/g tissue weight, one of the highest tissue concentrations known. The FAD is probably associated with an unidentified flavoprotein concentrated in the basal, synaptic region of the hair cell.

Acoustic Maculae↗
PubMed2006

Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyase.

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD(ox). Photolyase reconstituted with FAD(ox) had about 34% activity of native enzyme, which increased to 83% when FAD(ox) was reduced to FADH(-). Reconstitution kinetics performed at 20 degrees C showed that apo-photolyase associates with FADH(-) much faster (k(obs) approximately 3,000 M(-1) s(-1)) than with FAD(ox) (k(obs)=16 [corrected] M(-1) s(-1)). The dissociation constant of the photolyase-FAD(ox) complex is about 2.3 microM and that of E-FADH(-) is not higher than 20 nM (pH 7.2).

Apoenzymes↗
PubMed2005

Quantitation of nicotinamide and serotonin derivatives and detection of flavins in neuronal extracts using capillary electrophoresis with multiphoton-excited fluorescence.

Capillary electrophoresis (CE) with multiphoton-excited fluorescence detection (CE-MPE) allows low-background analysis of spectrally distinct fluorophores using a single long-wavelength laser. Extracts were prepared from immortalized rat raphe nuclei neurons, and were analyzed by CE-MPE. Native fluorescence was detected from reduced nicotinamide adenine dinucleotide (NADH) and its phosphorylated form (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), riboflavin, serotonin, and 5-hydroxytryptophan (5HTrp). Quantitation of exogenous serotonin (taken up by cells) and endogenous NADH and 5HTrp was possible using internal standards or standard addition. This system should be useful to study monamine oxidase inhibitors (MAOIs) and selective serotonin reuptake inhibitors (SSRIs).

Electrophoresis, Capillary↗
PubMed2005

Histone demethylation catalysed by LSD1 is a flavin-dependent oxidative process.

Lysine-specific histone demethylase 1 (LSD1) is a very recently discovered enzyme which specifically removes methyl groups from Lys4 of histone 3. We have addressed the functional properties of the protein demonstrating that histone demethylation involves the flavin-catalysed oxidation of the methylated lysine. The nature of the substrate that acts as the electron acceptor required to complete the catalytic cycle was investigated. LSD1 converts oxygen to hydrogen peroxide although this reactivity is not as pronounced as that of other flavin-dependent oxidases. Our findings raise the possibility that in vivo LSD1 might not necessarily function as an oxidase, but it might use alternative electron acceptors.

Catalysis↗
PubMed2006

Identification of a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase.

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.

Dithionitrobenzoic Acid↗
PubMed1998

1,4-dihydro-2,3-quinoxalinediones as potential flavin metabolites and excitatory amino acid receptor ligands. Part 1: Synthesis and pharmacological evaluation of the benzylic oxidation series of 1,4-dihydro-6,7-dimethyl-2,3-quinoxalinedione.

A series of five 6,7-disubstituted 1,4-dihydro-2,3-quinoxalinediones was prepared, two of which are known microbial flavin metabolites and three of which are potential flavin metabolites. Four of the five compounds inhibited specific binding of [3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid ([3H]AMPA), [3H]kainic acid, and [3H]6-cyano-1,4-dihydro-7-nitro-2,3-quinoxalinedione ([3H]CNQX) in rat brain homogenate fractions, with IC50 values in the low micromolar range (the fifth compound competed only with [3H]CNQX). Two of the compounds were moderately potent AMPA antagonists in an in vitro functional test.

Animals↗
PubMed2003

Crystal structure of halophilic dodecin: a novel, dodecameric flavin binding protein from Halobacterium salinarum.

A novel, 68 amino acid long flavoprotein called dodecin has been discovered in the proteome of Halobacterium salinarum by inverse structural genomics. The 1.7 A crystal structure of this protein shows a dodecameric, hollow sphere-like arrangement of the protein subunits. Unlike other known flavoproteins, which bind only monomeric flavin cofactors, the structure of the dodecin oligomer comprises six riboflavin dimers. The dimerization of these riboflavins along the re-faces is mediated by aromatic, antiparallel pi staggering of their isoalloxazine moieties. A unique aromatic tetrade is formed by further sandwiching of the riboflavin dimers between the indole groups of two symmetry-related Trp36s. So far, the dodecins represent the smallest known flavoproteins. Based on the structure and the wide spread occurrences in pathogenic and soil eubacteria, a function in flavin storage or protection against radical or oxygenic stress is suggested for the dodecins.

Amino Acid Sequence↗
PubMedDate not supplied

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles.

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.

Dose-Response Relationship, Drug↗
PubMed1995

Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415.

The heme- and flavin-binding domains of constitutive rat neuronal nitric oxide synthase (NOS) were expressed in Escherichia coli as distinct polypeptides with properties characteristic of the intact enzyme. The amino-terminal heme-binding domain (residues 1-714) was expressed using the expression vector pCW. The denatured molecular mass of the expressed protein was 80 kDa, and the protein was shown to be immunoreactive to rabbit anti-NOS IgG. The NOS hemoprotein exhibited a ferrous-carbon monoxide difference spectrum with a wavelength maximum at 445 nm. Spectral perturbation with L-arginine and BH4 elicited a type I difference spectrum, confirming the presence of binding sites for these molecules within the N-terminal NOS polypeptide. Site-directed mutagenesis was applied to the putative axial heme ligand, cysteine-415, generating the histidine mutant, which confirmed the identity of the proximal ligand. NOS flavoproteins, with (C1, residues 715-1429) and without (C2, residues 749-1429) an amino-terminal calmodulin-binding motif, were expressed using the vector pPROK-1. The C1 and C2 flavoproteins were immunoreactive to anti-NOS IgG and were sized at approximately 80 kDa. Both of the purified flavoproteins exhibited optical absorbance properties typical of a flavin prosthetic group, with wavelength maxima at 380 and 450 nm, and were competent in NADPH-dependent electron transfer to cytochrome c, with observed rates of approximately 2-4 mumol/min/mg. The bacterial expression of the NO synthase heme-binding oxygenase and flavoprotein oxidoreductase domains as isolated proteins with specific properties of the intact enzyme represents an important development in structure-function studies of this complex enzyme.

Amino Acid Oxidoreductases↗