Isolation of 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine from a riboflavin-adenine-deficient mutant of Bacillus subtilis.

H Mitsuda,K Nakajima,Y Yamada

doi:10.1016/s0021-9258(17)38064-x

Abstract

Studies were carried out to determine possible intermediates involved in the biosynthetic pathway of riboflavin, using resting cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis AJ1988. The cells excreted 6,7-dimethyl-8-ribityllumazine, the end product in the biosynthetic pathway, into the incubation medium in large amounts. The addition of glyoxal caused a large accumulation of a green fluorescent compound; an inverse relation was observed between the formation of the lumazine and the concentration of glyoxal. Furthermore, added [2-14C]guanine effectively incorporated into the lumazine and the fluorescent compound in the same specific activity during incubation. The fluorescent compound was isolated, purified, and identified by paper chromatographic, fluorometric, and spectrophotometric analyses. It was proved to be 8-(1'-D-ribityl)lumazine, which appeared to have been formed by a reaction between glyoxal and a possible intermediate in the cells. Accordingly, 4-(1'-D-ribitylamino)-5-amino-2,6-dihydroxypyrimidine was concluded to be an immediate precursor of 6,7-dimethyl-8-ribityllumazine.

Highlights

Our results indicate that the compound isolated is 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine and that it may be an immediate precursor of 6,7-dimethyl-&ribityllumazine, which is the substrate for the riboflavin synthetase reaction [23, 24]
Studies on possible intermediates involved in flavinogenesis were made with resting cells of a riboflavin-adenine-deficient mutant, Bacillus subtilis AJ 1988
The mutant cells appear to lack riboflavin synthetase (EC 2.5.1.9) which catalyzes the conversion of 6,7-dimethyl-8-ribityllumazine to riboflavin [23, 24]

Summary

PROCEDURES

Organism -A riboflavin-adenine-deficient mutant, Bacillus subtilis AJ1988, was the gift of Ajinomoto Co. This process was repeated four times after which the combined fluorescent eluate was evaporated to dryness. The green fluorescent fractions were combined and directly run through a column (3.2 x 30 cm) of DEAE-cellulose (OH-), followed by washing and elution with the above solution These fractions were collected and dried in UQCUO, after which they were dissolved in 10 ml of distilled water. The 8-ribityllumazine fraction or the 6,7-dimethyl-8-ribityllumazine fraction was separately placed on a column (0.9 x 15 cm) of DEAE-cellulose (OH ), eluted with 8 rn~ acetic acid at an initial flow rate of 1 ml min. The green fluorescent compound was isolated from the resting cell medium containing 0.03% glyoxal which was incubated. In the calculation of the specific activity of 6,7-dimethyl-8-ribityllumazine, the values which were obtained by subtracting the amounts of 6,7dimethyl-8-ribityllumazine at the start of incubation from those at the 20th h, were used as the true amounts of the lumazine formed during incubation

RESULTS

Incorporation

DISCUSSION