Biosynthesis of vitamin B12. Some properties of the 5,6-dimethylbenzimidazole-forming system of Propionibacterium freudenreichii and Propionibacterium shermanii.

1. Homogenates of Propionibacterium freudenreichii transform riboflavin into 5,6-dimethylbenzimidazole. This process is stimulated by nicotinamide. Homogenates of Propionibacterium shermanii form only small amounts of 5,6-dimethylbenzimidazole from riboflavin in the absence of nicotinamide, but also form appreciable amounts in the presence of nicotinamide. 2. The stimulation of the 5,6-dimethylbenzimidazole-forming system by nicotinamide shows a lag phase which is abolished by preincubation of the homogenate with nicotinamide. Since no lag phase is observed when nicotinamide is replaced by nicotinate, nicotinate seems to be the true stimulating agent. These observations are in agreement with the fact that nicotinamide is rapidly split to nicotinate in homogenates of P. freudenreichii. 3. The 5,6-dimethylbenzimidazole-forming homogenate system is only active at a high buffer concentration (0.3--0.5 M) and in the presence of oxygen. The system has a pronounced oxygen optimum. 4. Flavin mononucleotide and flavin-adenine dinucleotide are better substrates for the 5,6-dimethylbenzimidazole-forming homogenate system than riboflavin. But with [1'-14C]riboflavin as substrate the specific radioactivity of 5,6-dimethylbenzimidazole is higher than the specific radioactivity of flavin--adenine dinucleotide and lower than the specific radioactivie substrate for the formation of 5,6-dimethylbenzimidazole. 5. A tentative reaction sequence for the transformation of flavin mononucleotide into 5,6-dimethylbenzimidazole is discussed.