BIOSYNTHESIS OF VITAMIN-B-12 IN ANAEROBIC-BACTERIA - EXPERIMENTS WITHEUBACTERIUM-LIMOSUM ON THE TRANSFORMATION OF 5-HYDROXY-6-METHYLBENZIMIDAZOLE, ITS NUCLEOSIDE, ITS COBAMIDE, AND OF 5-HYDROXYBENZIMIDAZOLYLCOBAMIDE IN VITAMIN-B-12

In anaerobic bacteria 5-hydroxybenzimidazole and 5-hydroxy-6-methylben zimidazole are precursors of the 5,6-dimethylbenzimidazole moiety of v itamin B-12. In order to elucidate the pathway from these bases to vit amin B-12, experiments on the transformation of 5-hydroxy-6-methylbenz imidazole, of droxy-6-methylbenzimidazole-alpha-D-ribofuranoside of 5- hydroxybenzimidazolylcobamide and of 5-hydroxy-6-methylbenzimidazolylc obamide into vitamin B-12 were carried out. The vitamin B-12 synthesiz ed by the anaerobe Eubacterium limosum in the presence of 5-hydroxy-6- methylbenzimidazole and L-[methyl-C-13]methionine was subjected to NMR spectroscopy. It revealed that the methyl group at C5 of the 5,6-dime thylbenzimidazole moiety was C-13 labeled, whereas the methyl group at C6 was unlabeled. This shows that the transformation of 5-hydroxy-6-m ethylbenzimidazole into the base moiety of vitamin B-12 occurs regiosp ecifically. droxy-6-methylbenzimidazole-alpha-D-ribofuranoside as well as 5 -hydroxybenzimidazolylcobamide and 5-hydroxy-6-methylbenzimidazo lylcobamide were also transformed into vitamin B-12 by E. limosum. Whe n 5-hydroxy-6-methylbenzimidazolylcobamide C-13 labeled at C2 of the b ase part and C-14 labeled in the ribose was used for this experiment, the vitamin B-12 obtained from this cobamide was C-13 and C-14 labeled in the same positions. This demonstrates that the alpha-glycosidic bo nd of the precursor cobamide is not split during the formation of vita min B-12. It can be deduced from these results that the precursor base s are transformed regiospecifically into their alpha-nucleotides, and partially into their cobamides. The alpha-nucleotides are then transfo rmed into alpha-ribazole-5'-phosphate and, subsequently, into vitamin B-12. Most likely the cobamides are degraded to the alpha-nucleotides before being used for the biosynthesis of vitamin B-12. A pathway for the latter process is suggested.