Mechanism of the ring contraction process in vitamin B₁₂ biosynthesis by the anaerobe Propionibacterium shermanii under aerobic conditions

Abstract

The mechanism of the ring contraction process during vitamin B12 biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with δ‐amino[1‐13C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and δ‐amino[1‐13C,1,1,4‐18O3]levulinic acid in combination with 13C‐NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin‐3x from formation of the γ‐lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon–oxygen bond at C1 of precorrin‐3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.

Metabolic pathways leading from amino acids to vitamin B12 in Propionibacterium shermanii, and the sources of the seven methyl carbons

“…Organism, chemicals and instruments Wet cells (450-500 g) of P. shermanii were cultivated and harvested as previously described [1,7]. A solution of l-[2-…”

“…13 C]aspartic acid, [2][3][4][5][6][7][8][9][10][11][12][13] C]glycine and l-[methyl- C]methionine generated via catabolism in the presence of THF (Figs 1 and 2A,B). The l-[methyl- (Figs 1 and 2C).…”

“…C]threonine, and [2][3][4][5][6][7][8][9][10][11][12][13] C]glycine added to the culture medium served mainly to enrich the seven methyl carbons of the corrin ring through C-methylation by S-adenosyl-l-[methyl- 13 C]methionine derived from catabolically generated l-[methyl- C]methionine, the 13 C-enrichment ratio of the methyl carbon at C-25 (the site of the first C-methylation) was less than those of the other six methyl carbons, probably due to the influence of endogenous d-glucose in P. shermanii. The almost identical 13 C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B 12 biosynthesis were completed before the amino acids were completely consumed.…”

“…The almost identical 13 C-enrichment ratios of the other six methyl carbons indicated that these C-methylations during vitamin B 12 biosynthesis were completed before the amino acids were completely consumed. However, in the case of l- [2][3][4][5][6][7][8][9][10][11][12][13] C]aspartic acid, the 13 C-enrichment ratios of five methyl carbons were low and similar, whereas the last two sites of C-methylation (C-53 and C-35) were not labeled, presumably because of complete consumption of the smaller amount of added label. The ratios of 13 C-incorporation into the seven methyl carbons are influenced by the conditions of amino acid feeding experiments in a manner that is dependent upon the order of C-methylation in the corrin ring of vitamin B 12 .…”

“…As glycine condenses with succinyl-CoA to produce ALA via the Shemin pathway in P. shermanii, and the methyl carbon of l-methionine is the biosynthetic origin of the seven methyl carbons of the corrin ring, feeding experiments with [2][3][4][5][6][7][8][9][10][11][12][13] C]glycine and l-[methyl- …”