Studies on the intermediates in the biosynthetic pathway of riboflavin. I. Identification of a green fluorescent compound, compound G1, accumulated in non-growing cells of Eremothecium ashbyii by the addition of dimeric diacetyl.

Abstract

Effects of sugars and carbonyl compounds (1%) on ribo-flavin formation were examined with non-growing cells of Eremothecium ashbyii. During non-growing cell incubation for 18hr, the addition of glucose markedly stimulated the formation of riboflavin, but the addition of pentoses (ribose, xylose, arabinose), ribitol and acetoin indicated no effect on the production. The supplementation of pyruvate, acetate, di-acetyl, glyoxal and acetoacetate, on the other hand, noticeably inhibited the formation of riboflavin. The effects of diacetyl monomer, dieter and trimer on the formation of riboflavin were further examined. Dimeric diacetyl (0.5%) among the diacetyl derivatives caused the accumulation of two green fluorescent compounds inside and outside of the cells during the 18hr incubation. The fraction (Compound G1), which was first eluted in Dowex 50W×4 column chromatography, was accumulated in large amounts indicating an inverse relation to riboflavin formation. Compound G1 was isolated from the mycelia and highly purified through many purification procedures. The structure of compound G1 was studied by paper chromatographic comparison, by determination of ultraviolet, emission, excitation and infrared spectra, by following the change of the maximum fluorescence of the reaction mixture containing the isolated compound in the presence of p-quinone and by examining the possibility of it as a substrate for riboflavin synthetase. The results obtained indi-cated that the isolated compound G1 is 6, 7-dimethyl-8-(1'-D-ribityl) lumazine. We speculated that dimeric diacetyl or its derivative becomes a significant inhibitor for the riboflavin synthetase reaction to result in the accumu-lation of 6, 7-dimethyl-8-ribityllumazine although the true inhibitor remains to be solved in future.