Biosynthesis of porphyrins and related macrocycles.: Part 51.: Proof that a reductive step occurs during the biosynthesis of vitamin B12 by the microaerophilic organism, Propionibacterium shermanii

5-Amino[4-C-13]laevulinic acid has been synthesised for enzymic conversion into C-13-labelled precorrin-2 12a. This was incubated with an enzyme system from Propionibacterium shermanii in the presence of [4-H-2(2)]NADH 5a and [4-H-2(2)]NADPH 6a to yield cobyrinic acid 15a, shown to carry H-2 at C-19 by appropriate C-13-NMR studies. Time same reducing cofactors but now stereospecifically labelled at C-4 with tritium were similarly used to biosynthesise cobyrinic acid which was H-3-labelled (15b) from the 4(R)-cofactors but carried no H-3 (15C) when the 4(S)-cofactors were used. Suitable degradation of the cobyrinic acid 15b after conversion into its ester 16b proved H-3-labelling at C-19. These results establish that the biosynthesis of vitamin B-12 in the microaerophilic organism Pr. shermaii involves a reductive Step in which a reductase enzyme transfers 4-H-R of the cofactor to C-19 of the macrocycle.