Nuclear Magnetic Resonance Studies of the Biosynthesis of Vitamin B12: THE ORIGIN OF THE METHYL GROUPS ON THE CORRIN RING

Abstract

Abstract The origin of the methyl group on carbon atom 1 of Ring A of the corrin ring of vitamin B12 was investigated by 13C and 1H magnetic resonance spectroscopy. Recently we found by the same technique that this methyl group does not originate from the 5-carbon atom of δ-aminolevulinic acid. This was of considerable interest, for it has been considered that in the fusion of Rings A and D the potential δ-methine bridge found in porphyrins, which does arise from δ-aminolevulinic acid, is converted to the methyl group found on carbon atom 1 of the corrin ring. Since the methyl group of methionine was previously found to be the source of at least six other methyl groups on the corrin ring, methionine was tested as a possible precursor of the methyl group on carbon atom 1. l-[methyl]-13C]Methionine was added to a growing culture of Propionibacterium shermanii and vitamin B12 was isolated. The 13C magnetic resonance spectra of the vitamin B12 in H2O, in 0.1 m KCN, and in aqueous HCl (pH l 1) showed seven methyl-13C resonances. Single frequency decoupling experiments enabled us to assign a 13C resonance to the C-1 methyl group. It appears, therefore, that in the synthesis of the corrin ring the aminomethyl group on Ring A or Ring D, which is the potential δ-methine bridge carbon atom found in the porphyrin ring, is eliminated and that carbon atom 1 (C-1) of the corrin ring accepts a methyl group provided by l-methionine. This finding should aid in the elucidation of the mechanism of synthesis of the functional isomers of vitamin B12 and of porphyrins. Analysis of our spectra also enabled us to make other resonance assignments and to differentiate between the relative positions in space of the two geminal methyl groups on carbon atom 12 of the corrin ring which arise by different mechanisms.