Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester

13C-labeled precorrin-6x is biosynthesized by cell-free protein preparations from Pseudomonas denitrificans in separate experiments using delta-amino[5-13C]levulinic acid and the corresponding delta-amino[4-13C]- and delta-amino[3-13C]levulinic acid-labeled forms in conjunction with S-[methyl-13C]adenosylmethionine for the latter two experiments. These labeled precorrin-6x samples, as their octamethyl esters, are studied by a range of NMR techniques. In addition, nuclear Overhauser effect difference measurements are made on unlabeled precorrin-6x ester to determine connectivities. The structure 6a so established for precorrin-6x ester (i) confirms the results reported in the preceding paper that precorrin-6x has a ring-contracted macrocycle, still carries the C-12 acetate residue, and stands at the oxidation level of a dehydrocorrin; (ii) reveals the unexpected methylation at C-11 not C-12, leading to a structure with separated chromophores; and (iii) implies that methyl migration from C-11 to C-12 occurs when precorrin-6x is converted into hydrogenobyrinic acid. Proposals for the biosynthesis of the corrin macrocycle of hydrogenobyrinic acid and vitamin B12 are made.