Riboflavin Synthesis Research Articles

Synthesis of a phosphorothioate analogue of flavin mononucleotide: reconstitution of the FMN-free form of NADPH-cytochrome P-450 reductase.

The chemical synthesis of riboflavin 5'-phosphorothioate (5'-FMNS) is described. 5'-FMNS is obtained from the alkaline hydrolysis of riboflavin 4',5'-cyclic phosphorothioate, which is produced upon reaction of riboflavin (RB) with thiophosphoryl chloride in trimethyl phosphate. 5'-FMNS has been tested for enzymatic reconstitution of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) depleted of its FMN prosthetic group, but containing its full complement (1 equiv) of FAD. The synthesis, purification, and characterization of 5'-FMNS are reported, and documentation of its efficacy in reconstituting the reductase by fluorometric and absorbance spectrophotometric measurements, as well as enzymatic activity, is presented. Data indicate that 5'-FMNS is totally competent in reconstituting NADPH-cytochrome c reductase activity, which requires the presence of both FAD and a flavin mononucleotide, and its fluorescence is completely quenched upon addition to FMN-free NADPH-cytochrome P-450 reductase.

Enzymatic synthesis of riboflavin and FMN specifically labeled with 13C in the xylene ring.

The condensation of 3-hydroximino-2-butanone (1) with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (2) yields 6,7-dimethyl-8-ribityllumazine (3). At slightly alkaline pH, the carbonyl group of 1 reacts preferentially with the 5-amino group of 2 (regioselectivity, 4:1). Under acidic conditions, the reaction occurs with higher yield and marginal regioselectivity of opposite direction (1:1.4). Appropriately 13C-labeled samples of 1 afford 3 labeled at C-6 alpha, C-6, C-7 or C-7 alpha. [6 alpha, 7 alpha-13C2]-3 was prepared by condensation of 2 with [1,4-13C2]diacetyl. The lumazines 3 were converted to riboflavin by the enzyme, riboflavin synthase, with almost quantitative yield. By this procedure, any C-atom of the carbocyclic moiety of riboflavin can be selectively labeled with 13C at high abundance. Phosphorylation yields the respectively 13C-labeled FMN samples.

Separation of epimericγ-aldonolactones in the synthesis of riboflavin

Separation of epimericγ-aldonolactones in the synthesis of riboflavin

44] Riboflavin synthetase from Eremothecium ashbyii and a salvage pathway of the by-product in the enzyme reaction

Publisher Summary This chapter discusses riboflavin synthetase obtained from Erernothecium ashbyii and a salvage pathway of the by-product in the enzyme reaction. In the pathway of riboflavin biosynthesis, purine with the exception of C-8 is incorporated into the isoalloxazine ring of riboflavin. Studies, with a guanine analog, 8-azaguanine and labeled purines in Eremothecium ashbyii, have revealed that the guanosine nucleotides, guanosine triphosphate (GTP) is the most immediate nucleotide precursor of riboflavin. An intermediate positioned after GTP in the biosynthetic pathway is believed to be 4-ribitylamino-2,5-diamino-6-hydroxypyrimidine, isolated from riboflavin-deficient mutants of yeast. Another intermediate, the nearest precursor to 6,7-dimethyl-8-ribityllumazine, was isolated from E. ashbyii and the mutants of yeast and Bacillus subtilis , and was identified as 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine that reacts with the 4-carbon compound to form the lumazine. The pyrimidine is structurally identical to the by-product in the riboflavin synthetase reaction, in which 2 mol of 6,7-dimethyl-8-ribityllumazine are consumed to form 1 mol of riboflavin and 1 mol of the by-product. The by-product was expected to be reutilized for the in vivo synthesis of riboflavin through the supplementation of the suitable 4-carbon compound to form 6,7-dimethyl-8-ribityllumazine. The experiments, with the riboflavin synthetase from E. ashbyii and a 4-carbon compound, diacetyl, present supporting evidence for the possibility.

Biosynthesis of Riboflavin in Vivo Isotope Incorporation Studies in Pichia guilliermondii Yeast

Summary In the riboflavin overproducing yeast Pichia guilliermondii (iron-deficient growth medium) labelling pattern of riboflavin and GMP isolated from RNA were determined after feeding D-[1-14C]ribose and D-[1-14C]ribitol. Distribution of radiocarbon over the carbon atoms of the ribityl side-chain of riboflavin and the ribose moiety of GMP was almost identical indicating a flow of radiocarbon to riboflavin via a common purine intermediate of riboflavin biosynthesis and nucleic acid formation. D-Ribose and ribitol are presumably consumed via D-ribose-5-phosphate in the nucleotide synthesis, thus sharing also to the riboflavin biosynthetic pathway. Data support the view that GTP is the immediate purine precursor of riboflavin synthesis. Interestingly, the carbon atom 1 of ribose seems to be specifically involved in the formation of the dimethylbenzene ring of riboflavin, too.

Intracellular accumulation of S-adenosylmethionine in a high-riboflavinogenic Eremothecium ashbyii grown in the presence of 3-amino-1,2,4-triazole

S-Adenosylmethionine accumulated in a high-riboflavinogenic Eremothecium ashbyii when grown in the presence of 3-amino-1,2,4-triazole, a specific inhibitor of riboflavin synthesis. The accumulated substance was extracted from the mycelia with HClO 4, and purified by the column chromatography on Dowex 1 × 2 and 50W × 8. Its structure was determined on the basis of ultraviolet spectra, behavior on paper chromatography, and hydrolysis experiments.

Syntheses of isoalloxazines and isoalloxazine 5-oxides. A new synthesis of riboflavin.

Syntheses of isoalloxazines and isoalloxazine 5-oxides. A new synthesis of riboflavin.

8-azaguanine and flavinogenesis in Eremothecium ashbyii

8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent). That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented. 8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis. Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.

Die Regulation der Synthese der Riboflavinsynthetase durch Eisen in flavinogenen Hefen

Summary The influence of iron on the synthesis of riboflavin (RF) and riboflavin synthetase (RFS) activity in the cells of some flavinogenic and non-flavinogenic yeasts was investigated. It is shown that during the cultivation of Pichia guilliermondii, Torulopsis candida and Schwanniomyces occidentalis in iron-deficient media the induction of RF oversynthesis is correlated with an increase in the RFS activity. This phenomenon did not occur in iron-deficient cultures of weak RF producers such as P. ohmeri, Candida utilis and C. pulcherrima. The incubation of iron-rich cells of T. candida with α,α′-bipyridine or o-phenanthroline chelaters which possess a high affinity to iron also causes a marked increase of RFS activity and the flavino-genesis rate. Cycloheximide in low concentration abolishes this effect; the same does amino-acid starvation of the cells of the methionine-deficient mutant of T. candida, MET-2. The addition of Fe (II) ions to the suspension of young iron-deficient cells prevents the increase of RFS activity. The Fe (II) ions and the autolysates of iron-rich cells do not inhibit the RFS activity in crude extracts of iron-deficient cells of this species. It is concluded that iron participates in the regulation of RFS synthesis in the cells of flavinogenic yeasts.

Zur Physiologie des Wachstums und der Riboflavin-Überproduktion von Eremothecium ashbyii: I. Der Einfluß von chemischen und physikalischen Faktoren

The life cycle of Eremothecium ashbyii may be divided into four phases: spore germination, growth of mycel, growth cessation associated with sporulation and riboflavin overproduction, and autolysis. The activity of respiration decreases with increasing age of culture and is not influenced by this phases. Using a white mutant strain we established, that the spore formation is not directly connected with riboflavin synthesis. The spores themselves do not produce riboflavin. The influence of various hydrocarbons, C-4-compounds, fatty acids, and surface agents on growth and riboflavin formation was investigated. Only such compounds stimulate the flavinogenesis, which contain oleic acid: sunflower oil, tween 80, and oleic acid itself. This effect bases likely on the surface activity of these compounds, because oleic acid is not used as sole carbon source. The cultivation temperature (20, 25, 30, 35 °C) influences growth and riboflavin overproduction in a different way. With an increase of temperature there is an increase of growth rate and a decrease of product formation rate. Der Kulturverlauf von Eremothecium ashbyii läßt sich in vier Abschnitte einteilen: Sporen-keimung, Mycelwachstum, Wachstunisstillstand mit Sporulation und Riboflavinproduktion, Autolyse. Die Atmung nimmt mit zunehmendem Alter der Kultur stark ab, sie bleibt von den Phasen unbeeinflußt. Mit Hilfe eines weißen Stammes wurde festgestellt, daß die Sporenbildung in keinem direkten Zusammenhang mit der Riboflavinsynthese steht, die Sporen selbst können kein Riboflavin bilden. Es wurde der Einfluß verschiedener Kohlenwasserstoffe, C-4-Verbindungen, Fettsäuren und Detergenzien auf Wachstum und Riboflavinbildung untersucht. Es stimulieren nur solche Verbindungen die Flavinogenese, die Ölsäure enthalten: Ölsäure selbst, Sonnenblumenöl, Tween 80. Da Ölsäure nicht als C-Quelle genutzt werden kann, beruht dieser Effekt wahrscheinlich auf der Herabsetzung der Oberflächenspannung. Die Züchtungstemperatur (20, 25, 30, 35°C) beeinflußt Wachstum und Riboflavinbildung in unterschiedlicher Weise. Während das Wachstum mit steigender Temperatur verbessert wird, sinkt die Produktbildungsrate. Bei 40°C wächst Eremothecium ashbyii nicht mehr.

Quantitative determination of isomeric intermediates of riboflavin synthesis, 3, 4-xylyl-6-phenylazo-and 3, 4-xylyl-2-phenylazo-d-ribitylamines, in industrial samples

Quantitative determination of isomeric intermediates of riboflavin synthesis, 3, 4-xylyl-6-phenylazo-and 3, 4-xylyl-2-phenylazo-d-ribitylamines, in industrial samples

The biosynthesis of pteridines. VI. Studies of the mechanism of riboflavin biosynthesis.

1 H N.m.r. spectroscopy has been used to study the deuterium exchange of the methyl protons in the lumazine precursor (6,7-dimethyl-8-D-ribitylpteridine-2,4-dione) of riboflavin, and in related compounds. The protons of the 7-methyl group exchange with the solvent, whereas those of the 6-methyl group do not. An explanation which involves a highly delocalized anionic species is suggested.One of the labelled lumazines has been converted into the corresponding flavin by an in vitro reaction which closely simulates the biosynthesis of riboflavin. The distribution of deuterium label in the flavin has been established, and a mechanism for its formation is proposed. The relevance of the results to the mechanism of the enzymic synthesis of riboflavin is discussed.

Intraspecific transduction in Staphylococcus epidermidis and interspecific transduction between Staphylococcus aureus and Staphylococcus epidermidis.

Phages were isolated from Staphylococcus epidermidis after exposure of cultures to mitomycin C. Using certain of these phages, genes controlling resistance to novobiocin, streptomycin, and erythromycin and genes involved in the synthesis of adenine and riboflavin were transferred from donor to recipient strains of S. epidermidis. Interspecific transduction of the Novr marker was accomplished between S. epidermidis and S. aureus. Attempts to transfer penicillin resistance were not successful.

Nondisplacement of Rat Tissue Riboflavin by 7-Chloro-8-methyl-flavin and the Stimulation of Intestinal Synthesis of Riboflavin

A study has been made of several factors involved in the mechanism by which 7-chloro-8-methyl-flavin produces a response in the riboflavin-deficient rat which is, for an extended period of time, indistinguishable from the response produced by the administration of riboflavin. When the riboflavin-deficient rat is given 7-chloro-8-methyl-flavin, it provides him with a stimulus which is mistaken as the stimulus he would receive from riboflavin. The animal increases his food consumption immediately, begins to grow, and begins to recover from the signs of deficiency. The ingestion of the additional food causes the intestinal bacteria to synthesize more riboflavin than is characteristic of the animal receiving no flavin. The additional riboflavin is insufficient to be solely responsible for the rapidity and the extent of the reversal of the deficiency state. Therefore, the apparent recovery of the animal must be due to a combination of the analog, a small additional quantity of riboflavin, and the consumption of food. Despite this apparent improvement in the animal’s general health, it cannot avoid the lethal results of the administration of the antiriboflavin analog. A small portion of the radioactive riboflavin administered to the rat is excreted as carbon dioxide.

The biosynthesis of pteridines. V. The synthesis of riboflavin from pteridine precursors.

The synthesis and reactions of various 8-substituted lumazine derivatives are described. In particular, it is shown that these compounds readily undergo hydration, and that the resulting carbinol-amines can take part in reactions which involve ring-opening in the pyrazine ring. These studies are extended to demonstrate a new chemical synthesis of riboflavin and related isoalloxazines from 8-substituted pteridine derivatives. A possible mechanism for this reaction, which is analogous to that involved in the biosynthesis of riboflavin, is discussed.Similar reactions of 2-iminopteridine derivatives are described.

BIOSYNTHESIS OF RIBOFLAVIN (VIT. B2) BY EREMOTHECIUM ASHBYII. IV. THE NUTRITIONAL REQUIREMENTS OF CARBON AND NITROGEN FOR E. ASHBYII.

1. The cis-forms of higher, unsaturated fatty acids, oleic and erucic markedly stimulated riboflavin synthesis while the transforms, elaidic and brassidic, had no effect. C8−C12 saturated fatty acids completely arrested the growth. 2. Some, locally available, oils showed some stimulation to riboflavin synthesis while showing a greater stimulation to growth. However the hydrolysates of these oils were either completely inhibitive to, ineffective upon, or slightly stimulatory to riboflavin synthesis. 3. The stimulation of oleic acid of the hydrolysates of olive oil depended upon the preservation of its degree of unsaturation. 4. The strong stimulation, caused by the nonsap. materials of lanolin, accounts for almost all the stimulation caused by the hydrolysates of this fat. However, cholesterol is not the nonsap. material responsible for this stimulation. 5. Some surface active agents such as lecithin, and saponin stimulated the vitamin formation. Of the detergents tested only tween 80 and, to a lesser extent, tween 60 showed a marked increase in the riboflavin yield in the shake cultures.

The physiology of riboflavin production by Eremothecium ashbyi.

SUMMARY: During the submerged batch cultivation of a riboflavin-producing strain of Eremothecium ashbyi three phases were observed. The first phase was characterized by rapid growth of mycelium, rapid utilization and oxidation of glucose and a decrease of pH value caused by accumulation of pyruvic acid. Subsequently acetoin accumulated in the medium. Glucose was oxidized incompletely since only 1.8 μmole oxygen were consumed/ μmole glucose utilized. The end of this phase was marked by exhaustion of glucose and cessation of growth. The second phase began with sporulation and was characterized by rapid synthesis of cell-bound riboflavin. Simultaneously a rapid increase in catalase activity and decreases of pyruvate and acetoin were observed. This was accompanied by a marked decrease in Qo2 on glucose while the Qo2 on pyruvate, threonine or acetaldehyde increased to a maximum. Ammonia accumulated in the medium and alkaline pH values were reached. The third phase was characterized by autolysis of mycelium which led to the release of riboflavin and to a decrease of enzymic activities. A comparison of all important physiological parameters was made with two strains of E. ashbyi of different riboflavin productivity. On the basis of correlation between riboflavin formation, catalase activity and respiration on acetaldehyde, an hypothesis is proposed to explain over-production of riboflavin by a shift from the initial eytochrome type of terminal respiration to the flavoprotein type which is, however, accompanied by over-production of the flavin prosthetic group.

BIOGENESIS OF RIBOFLAVIN IN GREEN LEAVES

1. V compound was produced non-enzymatically from G compound.2. The conversion reaction of G compound into V compound proceeds enzymatically in the presence of the enzyme preparation obtained from the spinach.3. The enzymatic formation of V compound from G compound requires the presence of molecular oxygen, and inhibited completely under anaerobic condition.4. The enzymatic reaction to form riboflavin from G compound proceeds without molecular oxygen.5. The stoichiometry of the enzymatic synthesis of riboflavin was established by the reaction under “anaerobic condition”.6. Formation of V compound was inhibited by KCN and KF.

The Influence of Diet Composition on the Intestinal Synthesis of Riboflavin and Intestinal Microflora (Part 5)

We have previously reported that sorbitol might have a riboflavin sparing action in the rat. Therefore, it seems, very interesting to investigate the mechanism of sparing action of riboflavin.In this report, the riboflavin content in cecum of rats supplemented with sorbitol was measured. Further, the changes that may occur in the intestine by sorbitol supplementation were also investigated.Growing rats of approximately 60-80 grams body weight were divided into two groups. The one was the riboflavin supplemented group, and the other was the riboflavin non-supplemented group.The former was administered orally 5γ of riboflavin per day per rat in addition to riboflavin contained in the diet. The latter was supplied only approximately 0.7γ of riboflavin per day per rat from the diet. Each group was divided into three diet groups: sucrose, sucrose plus sorbitol and the dextrin group.Riboflavin content and bacterial counts in cecum were estimated by the same method as described in the previous report.It was found that, the similar results as in weight gain were also obtained in total riboflavin content of cecum.Moreover, no significant differences were seen in cecal total bacterial counts. However, the increase in coli form bacterial count in cecum was observed in the sucrose diet group, while coli form bacteria had a tendency to decrease in the sucrose plus sorbitol diet.From these results, it has been further asertained that sorbitol possesses a riboflavin sparing action. Moreover, it seems that coli form bacteria does not contribute so much to riboflavin synthesis in rat intestine.

Intestinal Synthesis of Riboflavin in the Rat

Intestinal Synthesis of Riboflavin in the Rat