Yeast flavin mononucleotide

Flavin mononucleotide was first isolated from the yellow en2yme in yeast by Warburg and Christian in 1932 (4). The yellow en2yme was spHt into the protein and the yellow prosthetic group (coen2yme) by dialysis under acidic conditions. Flavin mononucleotide was isolated as its crystalline calcium salt and shown to be riboflavin-5Lphosphate its stmeture was confirmed by chemical synthesis by Kuhn and Rudy (94). It is commercially available as the monosodium salt dihydrate [6184-17 /, with a water solubiUty of more than 200 times that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubili2ers needed for riboflavin. 

Once the hexameric structure of the yeast FAS was established, the number of functional active sites still remained to be determined. Earlier studies had shown that the functional complex contains approximately six equivalents each of two prosthetic groups 4 -phosphopantetheine [60,63], necessary for the AGP functionality, and flavin mononucleotide [64], an essential component of the enoyl reductase activity. These studies provided an early indication that each of the six active sites in the complex has a full set of the chemical groups necessary for fatty acid synthesis. Nevertheless, conflicting reports appeared in the literature as to the competence of six active sites. Whereas some reports suggested the possibility of half-sites reactivity (only three of the six sites are catalytically competent) [65, 66], others proposed that all six active sites could synthesize fatty acids [62]. Subsequent active site titration experiments were performed which quantitated the amount of fatty acyl products formed in the absence of turnover [67]. Single-turnover conditions were achieved through the use of... 

In 1915, Harden and Norris observed that dried yeast, when mixed with lactic acid, reduced methylene blue and formed pyruvic acid 4). Thirteen years later Bernheim prepared an extract from acetone-dried baker s yeast, which had lactate dehydrogenase activity (5). Bach and co-workers demonstrated that the lactate dehydrogenase activity was associated with a 6-type cytochrome, which they named cytochrome 62 (6). In 1954, the enzyme was crystallized, enabling the preparation of pure material and the identification of flavin mononucleotide as a second prosthetic group (2). Since then, significant advances have been made in the analysis of the structure and function of the enzyme. Much of the earlier work on flavocytochrome 62 has already been summarized in previous review articles (7-10). In this article we shall describe recent developments in the study of this enzyme, ranging fi om kinetic, spectroscopic, and structural data to the impact of recombinant DNA technology. 

Cytochrome 2 functions in yeasts both as an electron transport system and as lactate dehydrogenase, combining the functions of two different proteins—L-lactate dehydrogenase and cytochrome b—into a single catalytic unit. The molecular mass of the enzyme is approximately 235,000. It consists of four subunits, each containing a group of flavin mononucleotide (FMN) and heme. FMN can be reversibly separated from the protein. The absorption spectrum of 2 corresponds to a low-spin complex. It is assumed that the transfer of electrons from lactate proceeds through flavin to the heme. ... 

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]. 

TLC on silica gel 60 plates was used in various TLC solvent systems for both determination and identification of flavin derivatives in baker s yeast and foods (plain yogurt and bioyogurt, raw egg white, and egg powder). The Rf values of two unknown compounds found in plain yogurt were identical to those of 7a-hydroxyriboflavin (Rf values 0.32 and 0.21) and riboflavin-p-galactoside (Rf values 0.14 and 0.10), but not to those of other flavin compounds [flavin adenine dinucleotide or FAD (Rf values 0 and 0), flavine mononucleotide or FMN (Rf values 0 and 0.05), 10-hydroxyethylflavin (/ f values 0.71 and 0.40), riboflavin (Rf values 0.55 and 0.32), and 10-formyhnethylflavin (Rf values 0.86 and 0.76)] by TLC on silica gel with chloroform-methanol-ethyl acetate (5 5 2) and 1-butanol—benzyl alcohol—glacial acetic acid (8 4 3) as solvents, respectively. ... 

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. 

Flavin mononucleotide (riboflavin-5 -phosphate) is synthesized from riboflavin with a purified enzyme from yeast according to the following equation ... 

FAD is similar in structure to DPN. It differs in that riboflavin replaces the nicotinamide riboside moiety. It is formed when red blood cells are incubated with riboflavin.A purified enzyme system from yeast catalyzes the synthesis of this coenzyme from ATP and flavin mononucleotide. ... 

Yeast mitochondrial flavocytochrome 2 (lactate cytochrome c oxido-reductase) catalyzes the transfer of electrons from L-lactate to various acceptors, cytochrome c being the physiological acceptor. The protoheme and flavin mononucleotide, because of their higher redox potential, can both be reduced completely by L-lactate the enzyme accepts a total of three electrons per protomer, which amounts to twelve electrons for the stable active tetramer. Both types of prosthetic group are quantitatively reoxidized by external acceptors. 

Peptone and yeast extract were purchased from Kyokuto Pharmaceutical Co, Ltd. (Tokyo, Japan). Riboflavin and riboflavin phosphate (flavin mononucleotide, FMN) were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). Lecithin from egg was purchased from Wako Pure Chemical Co. (Osaka, Japan). Other reagents were commercially available analytical... 

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