Biosynthesis of porphyrins and related macrocycles. Part 51.1,2 Proof that a reductive step occurs during the biosynthesis of vitamin B12 by the microaerophilic organism, Propionibacterium shermanii

Abstract

5-Amino[4-¹³C]laevulinic acid has been synthesised for enzymic conversion into ¹³C-labelled precorrin-2 12a. This was incubated with an enzyme system from Propionibacterium shermanii in the presence of [4-²H₂]NADH 5a and [4-²H₂]NADPH 6a to yield cobyrinic acid 15a, shown to carry ²H at C-19 by appropriate ¹³C-NMR studies. The same reducing cofactors but now stereospecifically labelled at C-4 with tritium were similarly used to biosynthesise cobyrinic acid which was ³H-labelled (15b) from the 4(R)-cofactors but carried no ³H (15c) when the 4(S)-cofactors were used. Suitable degradation of the cobyrinic acid 15b after conversion into its ester 16b proved ³H-labelling at C-19. These results establish that the biosynthesis of vitamin B₁₂ in the microaerophilic organism Pr. shermanii involves a reductive step in which a reductase enzyme transfers 4-H_R of the cofactor to C-19 of the macrocycle.