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At least 73 records · Page 4Linked to original sources
PubMed2001

Artificial flavin receptors: effects of hydrogen bonding on redox properties of a flavin mimic.

This review describes the roles of hydrogen bonding on the redox properties of a flavin mimic by using artificial flavin receptors. The receptors exploited are melamine derivatives bearing guanidinium ion(s) that strongly bind 6-azaflavin through five or seven hydrogen bonds involving N(1), C(2)=O, N(3)-H, C(4)=O, N(5), and N(6) positions in CHCl3 and CHCl3-acetonitrile. It has been shown that receptors are quite useful for examination of the hydrogen bonding effects on the redox potential, stability of the anionic semiquinone radical, and the oxidation activity of 6-azaflavin. The functionalized receptors have a substrate- or a metal-binding site have been shown to facilitate the reactions by forming the noncovalent assemblies. A possibility as an apoprotein model of the receptors has been mentioned also.

Binding Sites↗
PubMed1982

A residue critical for flavin binding in flavocytochrome b2 from Baker's yeast. Inactivation and labeling of flavin-free enzyme by 2-keto-3-butynoate.

The reagent 2-keto-3-butynoic acid is the product formed in the reaction between the suicide reagent 2-hydroxy-3-butynoate and a number of flavoproteins. We describe in this paper the inactivation of flavin-free flavocytochrome b2 by 2-keto-3-butynoate, in a rapid reaction which introduces 0.9 mol reagent for total inactivation. The modification results in loss of affinity for flavin and affects a cysteine residue. We also describe in this paper a simple enzymatic method for preparing 2-keto-3-butynoate, as well as some properties of the reagent, in particular its stability and susceptibility to nucleophilic attack. We show that at neutral pH it is highly specific for thiol compounds. Some properties of the adduct formed with glutathione are described. These experiments should pave the way for the use of 2-keto-3-butynoate with other proteins.

Binding Sites↗
PubMed1999

Kinetics and thermodynamics of free flavins and the flavin-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol-gel-derived glass.

We report on the steady-state and time-resolved fluorescence from the redox active site flavine adenine dinucleotides (FADs) that are bound to glucose oxidase (GOx) when this enzyme is dissolved in aqueous solution or sequestered within a sol-gel-derived glass. To the best of our knowledge, this represents the first report on the actual dynamics of an enzyme active site when the enzyme is part of a sol-gel-derived glass. The results from these experiments show that the "free" FAD intramolecular folding/unfolding kinetics are slowed 3-10-fold within the glass vs solution. The intramolecular exciplex formation event (i.e., excited-state FAD residue folding/unfolding) is completely arrested for the GOx-bound FAD if the enzyme is sequestered within a glass in the absence of glucose. This is significantly different from the behavior of GOx dissolved in solution. However, despite this difference in behavior, the GOx molecules that are sequestered within the glasses continue to function somewhat like GOx dissolved in aqueous solution if they are challenged with glucose. We also found that the GOx molecules do not leach from the glass and they exhibit rotational mobility that is only 2-fold less than GOx dissolved in aqueous solution at 20 degrees C. In aqueous solution or within these glasses, the enzyme pocket that hosts the FAD redox sites opens up by 25-30% when GOx is challenged with glucose. Finally, we present preliminary analytical results for film-based sol-gel-derived biosensors that contain GOx, L-amino acid oxidase or cholesterol oxidase wherein the intrinsic FAD fluorescence produces the analytical signal.

Binding Sites↗
PubMed1996

Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution.

NfsB is an oxygen-insensitive nitroreductase of Escherichia coli with significant amino acid sequence homology to the major flavin reductase (FRase I) of Vibrio fischeri. Here, we show that NfsB is convertible to an FRase I-like flavin reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.

Amino Acid Sequence↗
PubMed1998

Conversion of NfsA, the major Escherichia coli nitroreductase, to a flavin reductase with an activity similar to that of Frp, a flavin reductase in Vibrio harveyi, by a single amino acid substitution.

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

Amino Acid Sequence↗
PubMed1986

31P- and 13C-NMR studies on the flavin-protein and flavin-ligand interactions in brewer's yeast old yellow enzyme.

The 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corresponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE(0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C]FMN or [4,10a-13C2]FMN, while apo OYE-I was reconstituted with [4a-13C]FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols.(ABSTRACT TRUNCATED AT 250 WORDS)

D-Amino-Acid Oxidase↗
PubMed1997

Flavin adenine dinucleotide and flavin mononucleotide metabolism in rat liver--the occurrence of FAD pyrophosphatase and FMN phosphohydrolase in isolated mitochondria.

In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD-->FMN and FMN-->riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification. Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover.

Acid Phosphatase↗
PubMed1999

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma by capillary electrophoresis and laser-induced fluorescence detection.

BACKGROUND: Riboflavin is the precursor of flavin mononucleotide (FMN) and FAD, which serve as cofactors for several redox enzymes. We have developed a capillary electrophoresis method for the determination of riboflavin and its two coenzyme forms in human plasma. METHODS: Trichloroacetic acid-treated plasma was subjected to solid-phase extraction on reversed-phase columns. The analytes were separated by micellar electrokinetic capillary chromatography in uncoated fused- silica capillaries filled with borate buffer containing 50 mmol/L sodium dodecyl sulfate, methanol, and N-methylformamide. Native fluorescence was monitored at 530 nm, using an argon laser operating at 488 nm as excitation source. RESULTS: The assay was linear over a concentration range of two orders of magnitude, and the limit of detection was far below physiological concentrations for all vitamers. The within-day and between-day coefficients of variation were 4-9% and 6-12%, respectively. The reference values (median, 5-95 percentiles) obtained by analyzing plasma from 63 healthy subjects were 8.6 nmol/L (2.7-42.5 nmol/L) for riboflavin, 7.0 nmol/L (3.5-13.3 nmol/L) for FMN, and 57.9 nmol/L (44.5-78.1 nmol/L) for FAD. CONCLUSIONS: Capillary electrophoresis with laser-induced fluorescence detection allows determination of all riboflavin vitamers far below physiological concentrations. The method may become a useful tool for the assessment of riboflavin status in humans.

Adult↗
PubMed2002

Riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in human plasma and erythrocytes at baseline and after low-dose riboflavin supplementation.

BACKGROUND: Vitamin B(2) exists in blood as riboflavin and its cofactors, flavin mononucleotide (FMN) and FAD. The erythrocyte glutathione reductase activation coefficient (EGRAC) has traditionally been used to assess vitamin B(2) status in humans. We investigated the relationships of EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD in elderly volunteers and their responses to riboflavin administration. METHODS: EGRAC and plasma and erythrocyte concentrations of riboflavin, FMN, and FAD were determined in 124 healthy individuals with a mean age of 69 years. The same measurements were made in a subgroup of 46 individuals with EGRAC > or =1.20 who participated in a randomized double-blind 12-week intervention study and received riboflavin (1.6 mg/day; n = 23) or placebo (n = 23). RESULTS: Median plasma concentrations were 10.5 nmol/L for riboflavin, 6.6 nmol/L for FMN, and 74 nmol/L for FAD. In erythrocytes, there were only trace amounts of riboflavin, whereas median FMN and FAD concentrations were 44 and 469 nmol/L, respectively. Erythrocyte FMN and FAD correlated with each other and with EGRAC and plasma riboflavin (P <0.05). All variables except plasma FAD responded significantly to riboflavin supplementation compared with placebo (P < or =0.04). The strongest increases were for riboflavin in plasma (83%) and for FMN in erythrocytes (87%). CONCLUSIONS: Concentrations of all B(2) vitamers except plasma FAD are potential indicators of vitamin B(2) status, and plasma riboflavin and erythrocyte FMN may be useful for the assessment of vitamin B(2) status in population studies.

Aged↗
PubMed1985

Site-specific immobilization of flavin adenine dinucleotide on indium/tin oxide electrodes through flavin adenine amino group.

A Mannich-type reaction was used to attach flavin adenine dinucleotide (FAD) covalently to aminosilane derivatized indium/tin oxide-coated glass plates. The aminosilane was activated with formaldehyde to give an intermediate that attached specifically to the adenine amino group of FAD. The presence of the intermediate also was demonstrated by coupling hydroquinone to the formaldehyde activated support. The immobilized FAD and hydroquinone were characterized by cyclic or differential pulse voltammetry. The immobilized FAD was shown to reduce the overpotential for NADH oxidation by 180 mV. In keeping with results for FAD on glassy carbon, FAD attached to indium/tin oxide at the adenine amino group did not lead to reconstitution of activity with apoglucose oxidase.

Apoenzymes↗
PubMed1994

Characterization of liver flavin-containing monooxygenase of the dogfish shark (Squalus acanthias) and partial purification of liver flavin-containing monooxygenase of the silky shark (Carcharhinus falciformis).

Flavin-containing monooxygenase (FMO) activity as N,N-dimethylaniline (DMA) N-oxygenation was characterized in microsomes from the smooth dogfish shark (Squalus acathias). DMA N-oxygenase activity from the liver of the dogfish shark was linear with increasing protein content and over 60 min. The optimal temperature for catalysis was 25 degrees C with a 76 percent reduction in activity when incubated at 15 degrees C and 99 percent loss of activity at 45 degrees C. Optimal pH was approximately 9.6. The maximum velocity for DMA N-oxygenase activity was calculated to be 1.3 nmol min-1 mg-1 with an apparent Michaelis constant of 44 microM. Methimazole oxidase activity was also observed in dogfish liver microsomes which was inhibited by trimethylamine (TMA). Inhibition of DMA N-oxygenase activity by TMA and thiobenzamide was competitive, while inhibition by methimazole was not competitive. Western blot analysis indicated a single liver protein from both Squalus and Carcharhinus of approximately 50 kDa that bound to antibodies raised against FMO 2. An attempt was made to purify FMO as methimazole oxidase from the liver of the silky shark. A single peak of about 10-fold purity was observed following passage through two chromatographic media (CM-Sepharose and HA-Agarose). However, no activity was recoverable after the FMO-containing fractions were applied to a 2'5' ADP-Sepharose column.

Animals↗
PubMed1978

Nitrogen-15 and carbon-13 nuclear magnetic resonance of reduced flavins. Comparative study with oxidized flavins.

Nitrogen-15 and carbon-13 nuclear magnetic resonance spectra of the fully reduced form of flavin were studied with riboflavin tetrabutyrate (RBUT), an organic solvent-soluble derivative of riboflavin. For the measurement of 15N resonances, 99% enriched [1,3-15N]RBUT and [1,3,5-15N]RBUT wwere synthesized. In order to assign the 13C resonances, 90% enriched [2-13C]RBUT, [4a-13C]RBUT, [4,10a-13C]RBUT, and [8-2H3]RBUT were employed. The upfield shift of N(5) resonance upon reduction was remarkable (286 ppm), while the N(1) signal moved only by 79 ppm. The one-bond 15N-H spin-spin coupling constant 1J[15N(5)-H] of the reduced RBUT was smaller than its 1J[15N(1)-H] and 1J[15N(3)-H]. These observations indicate that N(5) changed into sp3 hybridization upon reduction and lost the character of planar nitrogen. Most of the 13C nuclei of the reduced form resonated at higher field than did those of the oxidized form, which is well explained by the increase in pi-electron densities. Among the 13C resonances, the upfield shift of C(4a) was remarkable (32 ppm), which explains the reactivity of C(4a) in oxygen flavoprotein complexation. 13C--15N spin-spin coupling constants were obtained from the measurements of 13C magnetic resonance of 15N-enriched RBUT. The values of the one-bond 13C--15N coupling constants increased markedly with protonation at N(1) and N(5) upon reduction.

Flavins↗
PubMed1991

Flavins as potential antimalarials. 2. 3-Methyl-10-(substituted-phenyl) flavins.

A series of 3-methyl-10-(substituted-phenyl)flavins was prepared and tested for antimalarial activity against the lethal parasite Plasmodium vinckei in mice. Several of these analogues were found to be effective antimalarial agents. A quantitative structure-activity relationship study was undertaken with 44 analogues and no satisfactory relationship could be established.

Animals↗
PubMed1983

Raman spectra of flavin bound in flavodoxins and in other flavoproteins. Evidence for structural variations in the flavin-binding region.

The resonance coherent anti-Stokes Raman scattering (CARS) spectra for a number of flavoproteins are found to be fingerprints for the particular type of flavoprotein. One group studied were the bacterial flavodoxins: Desulfovibrio vulgaris, Desulfovibrio desulfuricans, Azotobacter vinelandii, Megasphaera elsdenii, Clostridium kluyverii and Clostridium formicoaceticum. The other examples were the enzymes lactate monooxygenase and glucose oxidase. FMN complexed to Vibrio harveyi luciferase, and a partially characterized non-fluorescent flavoprotein from Photobacterium leiognathi. In the frequency range 1700-1100 cm-1, differences in the frequency positions and relative intensities of the prominent bands are reflections of the interactions of the isoalloxazine ring with the protein. Based on tentative assignment of the vibrational modes in flavin models, the spectra are interpreted in terms of hydrogen bonding between the amino acid residues of the binding site and particular atoms of the isoalloxazine ring.

Bacterial Proteins↗
PubMed1976

[Free radicals in the flavin reduction reaction. Interaction of reduced nicotinamide adenine dinucleotide with flavin in solution].

The study of the flavin mononucleotide (FMN)-reduced nicotinamide adeninedinucleotide (NADH) reaction was carried out both under aerobic and anaerobic conditions, using spectrophotometric and titrimetric methods. The consumption of NADH was shown to exceed two times the consumption of FMN in the anaerobic reaction and the rate constant in the aerobic reaction was found to be about 4 times of that of the anaerobic reaction. Moreover, the replacement of anaerobic conditions by aerobic ones at pH 5.0 resulted in a considerable increase of proton consumption rate in the reaction course. The data obtained are contradictory to the generally accepted hypothesis of hydrid-ion transfer in the reaction of NADH oxidation. It was assumed that this reaction proceeded through a homolytic pathway.

Flavin Mononucleotide↗
PubMed2006

Insulin in flavin-containing monooxygenase regulation. Flavin-containing monooxygenase and cytochrome P450 activities in experimental diabetes.

Microsomal monooxygenases - cytochrome P450 (CYP, EC 1.14.14.1) and flavin-containing monooxygenase (FMO, EC 1.14.13.8) - have profound roles in drug metabolism. While the induction of some metabolic enzymes such as hepatic FMO and intestinal CYP1A, CYP2B is recognized in experimental diabetes, the effect of insulin treatment on FMO and intestinal CYP3A in diabetic animals has not been reported before. Changes in abundance and activity of hepatic and intestinal microsomal CYPs and FMO were studied in streptozotocin-induced diabetic rats either treated or not with insulin. Hepatic FMO1 activity increased in diabetic rats, but it was restored to control value on insulin treatment. Insulin itself had no effect on FMO1 activity in non-diabetic animals. A remarkable increase of total CYP content was accompanied by a reduced CYP3A specific enzyme activity in the small intestine of diabetic animals. The extent of these changes decreased on insulin treatment. Both, hepatic FMO1 and intestinal CYP3A activity correlated with average blood glucose concentration in untreated diabetic rats. These results indicate that insulin is involved in the regulation of hepatic FMO1 and intestinal CYP3A in rats. Blood glucose level is a good marker for FMO induction. The marked reduction of intestinal CYP3A capacity suggests that diabetes exerts a substantial effect on the activity of most determining intestinal CYP enzyme.

Animals↗