Flavin adenine dinucleotide, isolation

Riboflavin-5 -Adenosine Diphosphate. Riboflavin-5 -adenosine diphosphate [146-14-5] (flavin—adenine dinucleotide, FAD), C27H33N9O15P2 (2), mol wt 785.56, was first isolated in 1938 from the D-amino acid oxidase as its prosthetic group (95), where it was postulated to be... 

As early as 1908, Rosenthaler found in the ferment mixture of emulsin a u-oxynitrilase , which directed the addition of hydrocyanic acid (hydrogen cyanide) to benzaldehyde asymmetrically to give x-hydroxybenzeneacetonitrilc (mandelonitrile)9. This result was confirmed1 °, however, it was not until 1963 that Pfeil ct al. first isolated and characterized the enzyme (R)-oxyni-trilase [EC 4.1.2.101 from bitter almonds (Prunus amygdalus)1 12. The yellow-colored enzyme contains a flavin-adenine dinucleotide (FAD)11 and loses its activity by splitting off this prosthet-... 

LSDl, also known as BHCllO, is the first lysine specific demethylase that was discovered. It has been assigned to group I of lysine demethylases (KDMl) [90, 91]. LSDl contains an amine oxidase domain responsible of the enzymatic activity and has been isolated as a stable component from several histone modifying complexes. The enzymatic characterization of this protein revealed that FAD (flavine adenine dinucleotide) is required as a cofactor for the removal of the methyl group. Furthermore, LSDl requires a protonated nitrogen in order to initiate demethylation so that this enzyme is only able to demethylate mono- or dimethylated substrates but not trimethylated substrates [98, 99]. 

Selected entries from Methods in Enzymology [vol, page(s)] Determination of FMN and FAD by fluorescence titration with apoflavodoxin, 66, 217 purification of flavin-adenine dinucleotide and coenzyme A on p-acetoxymercurianiline-agarose, 66, 221 a convenient biosynthetic method for the preparation of radioactive flavin nucleotides using Clostridium kluyveri, 66, 227 isolation, chemical synthesis, and properties of roseoflavin, 66, 235 isolation, synthesis, and properties of 8-hydroxyflavins, 66, 241 structure, properties and determination of covalently bound flavins, 66, 253 a two-step chemical synthesis of lumiflavin, 66, 265 syntheses of 5-deazaflavins, 66, 267 preparation, characterization, and coenzymic properties of 5-carba-5-deaza and 1-... 

The hydroxynitrile lyase (HNL) class of enzymes, also referred to as oxynitrilases, consists of enzymes that catalyze the formation of chiral cyanohydrins by the stereospecific addition of hydrogen cyanide (HCN) to aldehydes and ketones (Scheme 19.36).275 279 These chiral cyanohydrins are versatile synthons, which can be further modified to prepare chiral a-hydroxy acids, a-hydroxy aldehydes and ketones, acyloins, vicinal diols, ethanolamines, and a- and P-amino acids, to name a few.280 Both (R)- and (.S )-selective HNLs have been isolated, usually from plant sources, where their natural substrates play a role in defense mechanisms of the plant through the release of HCN. In addition to there being HNLs with different stereo-preferences, two different classifications have been defined, based on whether the HNL contains a flavin adenine dinucleotide (FAD) co-factor. 

The enzymes discussed in this paragraph are D- and L-amino acid oxidases, the presence of which has been detected in numerous organisms and tissues. They were only recently identified in bacteria and the corresponding enzymes were isolated (for a review see [54]). These enzymes are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of amino acids (Scheme 13.21). 

As a result of collaborative work among the laboratories of Ehren-berg, Hemmerich, and Singer 161-163), the structure of the bound flavin was elucidated to be 8 -(A(-3-histidyl) flavin adenine dinucleotide. The structure of the histidyl flavin and the sequence of a flavin-bound penta-peptide isolated by proteolytic digestion are shown in Fig. 28. Singer et al. 23,25) have discussed the details of these studies. 

Xanthine oxidase, which is capable of catalyzing the conversion of hypoxanthine and xanthine to uric acid, was first detected in 1882 by Horbaczewski (Hll), who noted that extracts of various tissues could catalyze the conversion of xanthine to uric acid. A similar enzyme was detected in milk (M15). These enzymes contain a flavin-adenine dinucleotide prosthetic group (C9). As a result of the essential nature of the flavin-adenine dinucleotide portion of the enzjmie, a striking parallelism was seen between the riboflavin content of the diet and the xanthine oxidase concentration in tissues of growing rats (DIO). The enzyme contains molybdenum. That the molybdenum is contained in a functionally important component has been demonstrated by several workers (G13, T5). Totter and his associates injected labeled molybdate into a cow, and then isolated the enzyme from the milk to show that the proportion between the molybdenum and flavin remained constant at a value of 0.5. Corran et al. (C9) postulated that the xanthine oxidase of milk is identical with the xanthine oxidase of liver, but the protein portions of the enzyme appear to differ. 

MAO catalyzes the oxidative deamination of catecholamines, 5-hydroxytryptamine (serotonin), and other monoamines, both primary such as NE, and secondary such as EP. It is one of several oxidase-type enzymes whose coenzyme is the flavin-adenine-dinucleotide (FAD) covalently bound as a prosthetic group (Fig. 9-3). The isoalloxazine ring system is viewed as the catalytically functional component of the enzyme. In a narrow view N-5 and C-4a is where the redox reaction takes place (i.e., +H+, +le or -H+, -le), although the whole chromophoric N-5-C-4a-C-4-N-3-C-2-N-l region undoubtedly participates. Figure 9-3 is a proposed structure of MAO isolated from pig brain (Salach et al., 1976).4... 

The most common forms of vitamin B2 are riboflavin 5 -phosphate (FMN) and flavin adenine dinucleotide (FAD), which are best known for their participation as co-factors (ligands) to some of the enzymes involved in electron transfer chains. The ligand is usually coupled to enzymes through the phosphate moiety and therefore isolation from tissue can be achieved by either mild acid hydrolysis or by enzymatic digestion with an acid phosphatase. 

More recently Huang and Cavalieri (1979) have studied a similar enzyme in mitochondria isolated from spinach leaves. This enzyme had a pH optimum at 8.0-8.5 with a A , for proline of 28 mAf. The enzyme was linked to the mitochondrial electron transport system and MgCl2 and flavin adenine dinucleotide were required for maximal activity. The spinach oxidase also was inactive after Triton X-100 treatment. 

Vitamin B2 is riboflavin (ll.lOSe), which is utilised for the synthesis of coenzymes flavin mononucleotide (riboflavin monophosphate) (FMN), and flavin adenine dinucleotide (FAD) (11.110) and (11.28). Riboflavin was isolated from yeast in 1932 [33]. 

Microsomal cytochromes P450 are membrane-bound. They accept electrons from a microsomal NADPH-cytochrome reductase, containing flavin adenine dinucleotide and flavin mononucleotide. All cytochromes P450 metaboUsing drugs and xeno-biotica isolated so far belong to this class. 

A specific kinase, flavokinase, yields flavin mononucleotide in the presence of riboflavin and ATP, and probably magnesium. Schrecker and Kornberg [97] described an enzyme that catalyzes the synthesis of flavin adenine dinucleotide from flavin mononucleotide and ATP. The enzyme was isolated from yeast, and similar enzymes have been found in animal tissues. The enzyme is called flavin adenine dinucleotide pyrophosphorylase. 

NADH cytochrome c reductase was isolated from pigeon breast and pig heart muscle. The enzyme was shown to contain four atoms of iron per flavin molecule. NADH cytochrome c reductase, like succinic dehydrogenase, is a ferroflavoprotein. The ratio of iron to flavin is four. The enzyme contains sulfhydryl groups that can be titrated by classical methods, but their oxidation has no effect on the enzymatic activity. In contrast, the removal of the metal leads to a decrease in the ability of the enzyme to reduce cytochrome c. As for succinic dehydrogenase, the structure of the flavin in NADH cytochrome c reductase is not clear. It was demonstrated that it is not flavin mononucleotide, but the identity of the flavin component with flavin adenine dinucleotide is not established in fact, the flavin component differs from the classical FAD by its chromatographic properties and its behavior in enzymic assays. It is not known if it is a structural variation of the flavin nucleotide or if the nucleotide is conjugated to a peptide. 

Barile, M., Passarella, S., Bertoldi, A., and Quagliariello, E., 1993. Flavin adenine dinucleotide synthesis in isolated rat liver mitochondria caused by imported flavin mononucleotide. Archives of Biochemistry and Biophysics. 

Straub and Warburg and Christian found that the prosthetic group of d-amino acid oxidase was a flavin derivative, not identical with FMN. The latter group isolated the coenzyme and showed it to be flavin adenine-dinucleotide (FAD). 

More than 100 years ago a fluorescent compound was isolated first fi om whey, and later from different biological materials. When it Ijecame clear that the isolated yellow pigments, named lactochrome, ovoflavin, or lactoflavin, had a common structure, the new compound was named riboflavin (vitamin B2) (for historical review see 2). In the years between 1933 and 1935 the structure and the main chemical reactions of riboflavin were studied and the chemical synthesis was performed. Soon afterward, the coenzyme forms, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), were isolated in pure form, and the structures were determined. In the last 50 years many flavoproteins were isolated and their physicochemical properties were studied. Succinate dehydrogenase was the first enzyme found with the prosthetic group (FAD) covalently bound to the protein. About 20 flavoproteins are now known to contain covalently bound coenzyme (mainly via carbon atom 8a) (3). In mammalian tissue, the number of covalently bound flavoproteins appears to be limited. 

Enzyme purification was the first application of bioaffinity chromatography [23] and remains an important use of this technique. In this type of separation, ligands, such as enzyme inhibitors, coenzymes, or cofactors, are used to purify and separate enzymes [25]. For instance, in 1968 and the first report of "modem" affinity chromatography, Cuatrecasas, Wilchek, and Anfinsen employed specific enzyme inhibitors to selectively isolate enzymes [1,4]. A more recent example was the use of a support containing flavin mononucleotides for the purification of flavin adenine dinucleotide synthetase [26]. Other examples have included the use of mono-, di-, and triphosphate nucleotides for the purification of kinases and the use of nicotinamide adenine dinucleotide for the isolation of dehydrogenases [26,27]. 

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