Biosynthesis of vitamin B 12: Timing of the methylation steps between uro'gen III and cobyrinic acid

Abstract

The sequence of methylation between uro'gen III and cobyrinic acid has been defined by applying 13C pulse-labeling methods to a cell-free system from Propionibacterium shermanii. Feeding experiments using unenriched S-adenosyl methionine ( 12CH 3-SAM) followed by 13C-enriched SAM ( 13CH 3-SAM) (or vice versa) at various intervals caused differentiation in the 13C NMR signals of the SAM-derived methyl groups in cobyrinic acid (isolated as cobester). Unenriched uro'gen III and sirohydrochlorin as substrates led to cobyrinic acid containing seven and five enriched methyl groups, respectively, which on NMR analysis gave as a sequence of methylation C-2 > C-7 > C-20 > C-17 > C-12α > C-1 > C-5 ∼ C-15.