Presence Of FAD Research Articles

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.

Purification and characteriation of 3‐hydroxyphenylacetate 6‐hydroxylase: a novel FAD‐dependent monooxygenase from a Flavobacterium species

3-Hydroxyphenylacetate 6-hydroxylase was purified 70-fold from a Flavobacterium sp. grown upon phenylacetic acid as its sole carbon and energy source. The presence of FAD and dithiothreitol during purification is essential for high recovery of active enzyme. SDS/PAGE of purified enzyme reveals a single band with a minimum molecular mass of 63 kDa. Analytical gel-filtration, sedimentation-equilibrium and sedimentation-velocity experiments indicate that the purified enzyme exists in solution mainly as a dimer, containing 1 molecule non-covalently bound FAD/subunit. 3-Hydroxyphenylacetate 6-hydroxylase utilizes NADH and NADPH as external electron donors with similar efficiency. The enzyme shows a narrow substrate specificity. Only the primary substrate 3-hydroxyphenylacetate is hydroxylated efficiently, yielding 2,5-dihydroxyphenylacetate as a product. During turnover, the substrate analogues 3,4-dihydroxyphenylacetate and 4-hydroxyphenylacetate are partially hydroxylated, exclusively at the 6' (2') position. The physiological product 2,5-dihydroxyphenylacetate acts as an effector, strongly stimulating NAD(P)H oxidation. The activity of 3-hydroxyphenylacetate 6-hydroxylase is severely inhibited by chloride ions, competitive to the aromatic substrate. In the native state of enzyme, two sulfhydryl groups are accessible to 5,5'-dithiobis(2-nitrobenzoate). Titration with stoichiometric amounts of either 5,5'-dithiobis(2-nitrobenzoate) or mercurial reagents completely blocks enzyme activity. Inactivation by cysteine reagents is inhibited by the substrate 3-hydroxyphenylacetate. The original activity is fully restored by treatment of the modified enzyme with dithiothreitol. The N-terminal amino acid sequence of the enzyme lacks the consensus sequence GXGXXG, found at the N-termini of all flavin-dependent external monooxygenases sequenced so far. The amino acid composition of 3-hydroxyphenylacetate 6-hydroxylase is also presented.

A monoclonal antibody recognizing the FAD-binding site of 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody against 4-aminobenzoate hydroxylase (EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, has been produced by the fusion of BALB/c mouse spleen cells immunized with the denatured enzyme and P3x63Ag8U1 myeloma cells in order to locate and characterize the catalytic site of the enzyme. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated its apoenzyme. The immunoprecipitation was inhibited in the presence of FAD, and the monoclonal antibody competitively inhibited the binding of FAD to the apoenzyme. The monoclonal antibody, therefore, recognizes the FAD-binding site of 4-aminobenzoate hydroxylase. Interestingly, it was shown that the monoclonal antibody was cross-reactive with FAD-dependent enzymes such as salicylate hydroxylase (EC 1.14.13.1) and D-amino acid oxidase (EC 1.4.3.3), and that it was specific for the FAD-binding sites of these enzymes. This fact suggests that these FAD-dependent enzymes have immunologically similar structures on their FAD-binding sites.

Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography.

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Nitrate Reductase from Suspension Cultured Cells of Marchantia Polymorpha L.

Summary Nitrate reductase from suspension cultured cells of a bryophyte, Marchantia polymorpha L., was purified to 550-fold by a procedure involving blue-Sepharose affinity chromatography. The purest enzyme used NADPH as an electron donor if FAD was added to the reaction mixture, but did not use NADH even in the presence of FAD. The specific activity of the purest enzyme was 3.9 μmoles nitrite produced/min/mg protein. The purest fraction possessed three associated activities; FAD dependent NADPH-cytochrome c reductase, FADH 2 - and MVH-nitrate reductase. A study of the effects of inhibitors on the three activities demonstrated that the NADPH-nitrate reductase consisted of two functional parts. The molecular weight of the intact enzyme determined by gel filtration was 220,000.

Influence de la polymérisation de la d‐aminoacide oxidase sur le comportement de l'enzyme immobilisée sur chitosane par fixation covalente

D-Amino-acid oxidase is a flavoprotein using FAD as cofactor. The enzyme has been immobilized in the presence of FAD on a non-porous matrix: chitosan. This support is covalently bound to the enzyme with glutaraldehyde as cross-linking reagent. It is characterized by a good mechanical resistance to mechanical stirring. The enzymatic assays have been performed in batch reactor with D-phenylglycine as substrate by a spectrophotometric method which is based on the variation of the absorbance at 252 or 280 nm. The behaviour of the biocatalysts has been studied during repeated assays of 1 h at 25 degrees C in the absence of exogenous FAD. The experimental results have been compared with those obtained with the soluble enzyme tested in the presence or in the absence of FAD. The dependence of D-amino-acid oxidase on FAD concentration has been studied. Immobilized enzyme on chitosan appears to be less sensitive to the association-dissociation equilibrium of FAD. This property and the capacity of the enzyme to polymerize spontaneously in solution according to the experimental conditions have been established. The fact that the enzyme can exist in various oligomeric forms is of major importance because its catalytic expression is dependent of this phenomenon. The polymerization is known to be responsible for a decrease of the maximal rate V of the enzyme. It has also been shown that in the same way this decrease was accompanied by an improvement of the affinity of enzyme for substrates. Furthermore, the value of the dissociation constant of the apoenzyme-FAD complex is significantly smaller as the degree of polymerization is high. The conclusion is that the dissociation of the cofactor can be avoided if the immobilization step is carried out at high concentration of enzyme which is favourable to its polymerization.

Covalent flavinylation of 6-hydroxy-D-nicotine oxidase involves an energy-requiring process

E. coli cells harbouring the recombinant plasmid pDB222 with the 6-HDNO gene under the control of the tac-promotor were induced with IPTG to synthesize a high amount of 6-HDNO protein. Part of this protein was present as 6-HDNO apoenzyme. The proportion of 6-HDNO apoenzyme formed could be increased when the induction of 6-HDNO synthesis by IPTG was performed in the presence of the inhibitor diphenyleneiodonium. The 6-HDNO apoenzyme thus formed could be transformed into enzymatically active holoenzyme in the presence of FAD by a process requiring an energy-generating system consisting of ATP, phospho enolpyruvate and pyruvate kinase. This finding suggests that an enzymatic step(s) is (are) involved in the covalent flavinylation of 6-HDNO.

Enzymes metabolizing dimethylamine, trimethylamine and trimethylamineN-oxide in the yeastSporopachydermia cereanagrown on amines as sole nitrogen source

Sporopachydermia cereana, an ascosporogenous yeast, grew on dimethylamine, trimethylamine or trimethylamine N-oxide as sole nitrogen sources and produced mono-oxygenases for dimethylamine and trimethylamine that were significantly more stable than the corresponding enzymes found in Candida utilis. No trimethylamine mono-oxygenase activity was found in S. cereana grown on dimethylamine. In cells grown on trimethylamine N-oxide (but not on the other nitrogen sources), evidence for an enzyme metabolizing the N-oxide, possibly an aldolase, but more probably a reductase was obtained. All these activities showed a similar requirement for the presence of FAD or FMN in the extract buffer during isolation to retain activity. Amine mono-oxygenase activities showed a similar sensitivity to inhibitors, including proadifen hydrochloride and carbon monoxide as the corresponding enzymes in C. utilis. The trimethylamine N-oxide-dependent oxidation of NADH was more sensitive to inhibition by EDTA, N-ethylmaleimide and β-phenylethylamine than the mono-oxygenases, and less sensitive to KCN, and activity was significantly higher with NADPH than was observed with the 2 mono-oxygenases.

Purification and characterization of two isofunctional forms of NAD(P)H: quinone reductase from mouse liver.

NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed enzyme which promotes two-electron reductions of quinones and thereby protects cells against damage by reactive oxygen species generated during oxidative cycling of quinones and semiquinone radicals. Quinone reductase activity represents a minor component (about 0.006%) of mouse liver cytosolic proteins under basal (uninduced) conditions. Two isofunctional forms of this quinone reductase have been purified to homogeneity (1700-fold) in 30% yield from the liver cytosols of female CD-1 mice in which the enzymes were induced by administration of 2(3)-tert-butyl-4-hydroxyanisole. The purification involved ion exchange, hydrophobic, and affinity chromatographies. The two enzyme forms have been designated "hydrophilic" and "hydrophobic" based on the order of elution from phenyl-Sepharose. The more abundant hydrophilic form has been crystallized in the presence of FAD in the form of macroscopic tetragonal crystals. The two forms have similar isoelectric points (pI 9.2) and subunit molecular weights (Mr = 30,000) and probably exist as dimers in the native state. Purified preparations of the enzymes are equiactive with NADH and NADPH and show almost complete dependence on added FAD for catalytic activity. The Km values for FAD of the hydrophilic and hydrophobic forms are 2.72 and 1.72 nM, respectively. Their catalytic activities are the same and are remarkably high for nicotinamide nucleotide-linked dehydrogenases; maximum velocities (expressed per mg of pure enzyme) approach 4000 units/mg of protein under appropriate assay conditions. When menadione is the electron acceptor, the Km value for this quinone is very low (Km congruent to 2 microM). Both enzyme forms are potently inhibited by dicoumarol. Rabbit antisera against the hydrophilic quinone reductase precipitate quantitatively the entire quinone reductase activity of mouse liver cytosols obtained from animals maintained on a standard diet or those induced with 3-tert-butyl-4-hydroxyanisole. The quinone reductase activity of rat liver cytosols is also quantitatively precipitated by this antiserum.

Preliminary crystallographic X-ray data for an NAD(P)H:quinone reductase from mouse liver.

The hydrophilic form of NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) from mouse liver cytosol has been crystallized in the presence of FAD. X-ray diffraction analysis showed these crystals to be tetragonal and to belong to the I422 space group. The unit cell measured a = b = 246.5 A and c = 70.4 A. Since the subunit molecular weight is 30,000, the asymmetric unit probably contains two pairs of dimers.

Reversible inactivation of maize leaf nitrate reductase

Preincubation of maize leaves crude extracts with NADH resulted in a progressive accumulation of nitrite which mimicked a rapid and lineal activation of nitrate reductase. Nevertheless, in partially purified preparations it was found that preincubation at pH 8.8 with NADH promoted a gradual inactivation of nitrate reductase. At pH 7.5, the enzyme was not inactivated by the presence of NADH alone, but, with the simultaneous presence of a low concentration of cyanide, a fast inactivation took place. The NADH-cyanide-inactivated nitrate reductase remained inactive after removing the excess of NADH and cyanide by filtration through Sephadex G-25. However, it could be readily reactivated by incubation with ferricyanide or by a short exposure to light in the presence of FAD. Prolonged irradiation caused a progressive inactivation of the photoreactivated enzyme.

Dissociation of FAD from the NAD(P)H-Nitrate Reductase Complex from Ankistrodesmus braunii and Role of Flavin in Catalysis

Ankistrodesmus braunii NAD(P)H-nitrate reductase is a complex hemoflavomolybdoprotein composed by eight similar subunits. The flavin prosthetic group, identified as FAD, is essential for the NAD(P)H-dependent activities of the complex, and is located before the heme chromo- phore in the enzyme electron transport chain from reduced pyridine nucleotides to nitrate. Fluorescence studies indicate that nitrate reductase can dissociate about 80% of its FAD by incubation at room temperature, the flavin dissociation being followed by a parallel decrease of NADH-nitrate reductase activity. Dissociation of FAD from the protein is easily increased by dilution or prolonged dialysis of the enzyme preparations. However, exogenous FAD specifically prevents the dissociation of enzyme-bound flavin, and protects the NAD(P)H-dependent activities. The Km for FAD, as a protector of NADH-cytochrome c reductase activity, is 4 nᴍ. In addition, dithioerythritol also prevents the flavin dissociation, and therefore the presence of free sulphydryl groups in the FAD-domain is suggested. FAD-depleted nitrate reductase, obtained by several methods, is unable to recover its original activity when incubated in the presence of FAD alone or with thiols.

FAD-dependent malate dehydrogenase from [formula omitted] sp. strain Takeo: A possible role of phospholipid

Using a homogeneously purified FAD-dependent malate dehydrogenase, a phospholipid-requiring enzyme from the particulate fraction of Mycobacterium sp. strain Takeo, specific and near stoichiometric binding of FAD to the apoenzymecardiolipin complex, but not to the free apoenzyme, was demonstrated by sucrose density gradient centrifugation in the presence of FAD. The result strongly indicates that the FAD-binding site of the apoenzyme is formed by the protein-phospholipid interaction. This is also supported by the fact that the protective effect of FAD on the stability and on pCMB inhibition of the apoenzyme was observed only in the presence of cardiolipin.

Enzymatic Oxidation of Isethionate to Sulfoacetaldehyde in Bacterial Extract1

Isethionate degradation in a bacterial extract was shown by the isolation of enzymes and by identification of an intermediate to take place in two steps; dehydrogenation to sulfoacetaldehyde and desulfonation leading to the formation of sulfite and acetate. The enzyme responsible for isethionate oxidation in the presence of FAD was particulate in nature and a solubilized preparation obtained by extraction with buffer of low ionic strength had oxidizing activities against only isethionate and n-butanol among compounds tested. The enzyme was inhibited by thiol and carbonyl reagents.

Enzymatic synthesis of C40 carotenes by cell-free preparation from Halobacterium cutirubrum.

[14C]Mevalonate or (14C)isopentenyl pyrophosphate was found to be converted to transphytoene, trans-phytofluene, lycopene, and beta-carotene by a cell-free 270 000 X g supernatant fraction prepared from Halobacterium cutirubrum cells that were broken by manual grinding with glass beads. Incubations were done under N2 in the dark at 37 degrees C in 4 M NaCl in presence of FAD, NADP, and MgCl2; ATP was also added when mevalonate was the substrate. This system was also capable of converting trans-(14C)phytoene to beta-carotene via the intermediates trans-phytofluene, zeta-carotene, neurosporene, lycopene, and gamma-carotene. Each of these labelled intermediates on incubation separately with the same enzyme system was shown to be converted to the intermediates farther down the pathway. The results of this study show that the biosynthetic pathway for the formation of C40 carotenes in H. cutirubrum proceeds as follows: isopentenyl pyrophosphate leads to trans-phytoene leads to trans-phytofluene leads to zeta-carotene leads to neurosporene leads to lycopene leads to gamma-carotene leads to beta-carotene. This pathway differs from that in higher plants in that the cis isomers of phytoene and phytofluene are not on the main pathway of carotene biosynthesis, as they are in higher plants. Furthermore, trans-phytoene, which has not been reported to have any role in higher plants, appears to be the main intermediate in carotene biosynthesis in H. cutirubrum.

Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora

Evidence is presented which suggests that the NAD(P)H-cytochrome c reductase component of nitrate reductase is the main site of action of the inactivating enzyme. When tested on the nitrate reductase (NADH) from the maize root and scutella, the NADH-cytochrome c reductase was inactivated at a greater rate than was the FADH 2-nitrate reductase component. With the Neurospora nitrate reductase (NADPH) only the NADPH-cytochrome c reductase was inactivated. p-Chloromercuribenzoate at 50 μM, which gave almost complete inhibition of the NADH-cytochrome c reductase fraction of the maize nitrate reductase, had no marked effect on the action of the inactivating enzyme. A reversible inactivation of the maize nitrate reductase has been shown to occur during incubation with NAD(P)H. In contrast to the action of the inactivating enzyme, it is the FADH 2-nitrate reductase alone which is inactivated. No inactivation of the Neurospora nitrate reductase was produced by NAD(P)H alone and also in the presence of FAD. The lack of effect of the inactivating enzyme and NAD(P)H on the FADH 2-nitrate reductase of Neurospora suggests some difference in its structure or conformation from that of the maize enzyme. A low level of cyanide (0.4 μM) markedly enhanced the action of NAD(P)H on the maize enzyme. Cyanide at a higher level (6 μM) did give inactivation of the Neurospora nitrate reductase in the presence of NADPH and FAD. The maize nitrate reductase, when partially inactivated by NADH and cyanide, was not altered as a substrate for the inactivating enzyme. The maize root inactivating enzyme was also shown to inactivate the nitrate reductase (NADH) in the pea leaf. It had no affect on the nitrate reductase from either Pseudomonas denitrificans or Nitrobacter agilis.

Stimulation of brain aromatic alkylamine N-methyltransferase activity by FAD and methylcobalamin

We studied the effects of methylcobalamin alone and with various reducing systems (FADH 2, FAD or its analogues in combination with NADH or β-mercaptoethanol) on the activity of partially purified aromatic alkylamine N-methyltransferase (AANMT) from rat brain. As expected, the specific activity of AANMT in the presence of methylcobalamin alone was enhanced (125% of control). Surprisingly, in the presence of FAD alone (but not FADH 2 et cetera ) it was 175% of control; and int the presence of methylcobalamin plus FAD plus β-mercaptoethanol it reached 307% of control. Preliminary evidence suggests that FAD induces allosteric kinetics for the activated enzyme with respect to the methyl donor 5-methyltetrahydrofolic acid (5-MTHF).

Biosynthesis of folate derivatives in vitro

Cell-free extracts of 3-day-old pea cotyledons and root tips have been examined for ability to synthesize derivatives of tetrahydropteroylglutamic acid in reaction systems which included formaldehyde, serine and formate as precursors of one-carbon units. The reaction products were isolated by DEAE-cellulose chromatography and assayed microbiologically using Lactobacillus casei and Pediococcus cerevisiae. Synthesis of 5-methyltetrahydropteroylglutamic acid was stimulated by addition of NADPH and the presence of FAD in the extraction buffer. 10-Formyl and 5-formyltetrahydropteroylglutamates were formed in all reaction systems but omission of the one-carbon precursor reduced these syntheses. The presence of 5,10-methylenetetrahydropteroylglutamate reductase activity was confirmed by use of 14C-labelled derivatives. The oxidative reaction had a pH optimum of 6·4 and was not inhibited by additions of l-methionine to the reaction system. Cotyledon and root tip extracts also catalyzed the transmethylation of homocysteine utilizing methyl groups from 5-methyltetrahydropteroylmonoglutamate. The reaction was strongly inhibited by l-methionine in vitro.

Optical Activity and Conformation Studies of Pig Heart Lipoamide Dehydrogenase

Abstract Flavin adenine dinucleotide has been dissociated from pig heart lipoamide dehydrogenase in the presence of 1.5 m guanidine hydrochloride. Measurements of the circular dichroism on the apoenzyme show evidence of a large change in conformation, based on the molar ellipticity in the region 240 to 200 mµ. When FAD is removed from the holoenzyme in guanidine, but in the presence of approximately equimolar FMN, no change appears in the circular dichroism of the apoenzyme in the 200 mµ region. The ultraviolet absorption of the apoprotein indicates that no FMN remains bound to the apoenzyme. Either FMN stabilizes the protein in the presence of guanidine or, after the loss of FAD, promotes the return of a conformationally different species to the native form of the enzyme. Lipoamide dehydrogenase activity is reconstituted in the presence of FAD and dithiothreitol.

Enzymatic deiodination of diiodotyrosine; possible mediation by reduced flavin nucleotide.

The deiodination of L-DIT in vitro by thyroid and liver tissue was studied. Supplements of FAD stimulated deiodination in dilute thyroid homogenates and subcellular particulate preparations enriched with NADPH. Sodium dithionite (0.05M) stimulated deiodination in the absence of NADPH and its effect was also enhanced by FAD. The products of deiodination were the same in NADPH and dithionite-supplemented systems in the presence of FAD. The stimulating effect of FAD under these conditions was detectable at micromolar concentration, was not dependent upon light or oxygen, and appeared to be enzymatically mediated. The distributions of deiodinating activity supported by NADPH and by dithionite among thyroid subcellular fractions were similar, both being predominantly microsomal; NADPHdependent activity appeared more sensitive to heat and aging. The stimulating effect of dithionitedithionite could not be explained on the basis of reduction of endogenous NADP with formation of effective concentration of NADPH si...