Biosynthesis of vitamin B 12 in anaerobic bacteria

In anaerobic bacteria 5‐hydroxybenzimidazole and 5‐hydroxy‐6‐methylbenzimidazole are precursors of the 5,6‐dimethylbenzimidazole moiety of vitamin B 12 . In order to elucidate the pathway from these bases to vitamin B 12 , experiments on the transformation of 5‐hydroxy‐6‐methylbenzimidazole, of 5‐hydroxy‐6‐methylbenzimidazole‐α‐ D ‐ribofuranoside, of 5‐hydroxybenzimidazolylcobamide and of 5‐hydroxy‐6‐methylbenzimidazolylcobamide into vitamin B 12 were carried out. The vitamin B 12 synthesized by the anaerobe Eubacterium limosum in the presence of 5‐hydroxy‐6‐methylbenzimidazole and L ‐[methyl‐ 13 C]methionine was subjected to NMR spectroscopy. It revealed that the methyl group at C5 of the 5,6‐dimethylbenzimidazole moiety was 13 C labeled, whereas the methyl group at C6 was unlabeled. This shows that the transformation of 5‐hydroxy‐6‐methylbenzimidazole into the base moiety of vitamin B 12 occurs regiospecifically. 5‐Hydroxy‐6‐methylbenzimidazole‐α‐ D ‐ribofuranoside as well as 5‐hydroxybenzimidazolylcobamide and 5‐hydroxy‐6‐methylbenzimidazolylcobamide were also transformed into vitamin B 12 by E. limosum . When 5‐hydroxy‐6‐methylbenzimidazolylcobamide 13 C labeled at C2 of the base part and 14 C labeled in the ribose was used for this experiment, the vitamin B 12 obtained from this cobamide was 13 C and 14 C labeled in the same positions. This demonstrates that the α‐glycosidic bond of the precursor cobamide is not split during the formation of vitamin B 12 . It can be deduced from these results that the precursor bases are transformed regiospecifically into their α‐nucleotides, and partially into their cobamides. The α‐nucleotides are then transformed into αribazole‐5′‐phosphate and, subsequently, into vitamin B 12 . Most likely the cobamides are degraded to the α‐nucleotides before being used for the biosynthesis of vitamin B 12 . A pathway for the latter process is suggested.