Studies on the 4-carbon compound needed for the formation of the O-xylene ring of riboflavin.

The 4-carbon compound necessary for the formation of the o-xylene ring of riboflavin, was examined using labeled glucose and diacetyl in resting cells of Eremothecium ashbyii. The specific activity-time curves of riboflavin and lumichrome in the tracer experiments with [U-14C] glucose indicated that the specific activity of riboflavin during the incubation is much higher than that of its photolytic product, lumichrome. It is far above the level expected if the ribityl moiety of an intermediate, 4-ribitylamino-5-amino-2,6-dihydroxy-pyrimidine (RAADP), was to be utilized as the source of the 4-carbon unit on the o-xylene ring of riboflavin. It was further demonstrated that the specific activities of diacetyl and lumichrome at 10.5 h of the incubation and those of crystalline riboflavin and lumichrome at the later stage (20 h) of the incubation were identical respectively. Radioactive diacetyl furthermore proved to be likewise incorporated with a two fold dilution into riboflavin and lumichrome at 17 h of the incubation. The results reveal that it is not the ribityl fragment of RAADP bu the diacetyl that is the 4-carbon unit for the formation of the pyrazine ring of 6,7-dimethyl-8-ribityllumazine, that is, the o-xylene ring of riboflavin in the mold of Eremothecium ashbyii, although we still do not know whether the diketone requiring reaction is enzymatic or non-enzymatic.