Nuclear magnetic resonance studies of the biosynthesis of vitamin B12. The origin of the methyl groups on the corrin ring.
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Biomedical subjects
Publications and source records attributed to D Shemin.
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delta-Aminolevulinic acid dehydratase from Rhodopseudomonas spheroides was covalently linked to Sepharose 4B, which had been activated with cyanogen bromide. A column containing this enzyme gel readily catalyzed the synthesis of the pyrrole porphobilinogen on continuous passage of a solution of delta-aminolevulinic acid. Under the conditions of the procedures, product inhibition was minimized and a 50 to 94 percent yield was attained. A column containing about 1 milligram of enzyme was continuously operated for 27 days. Although its total activity appeared to be reduced about 30 percent at the end of this time, the bound enzyme produced approximately 200 milligrams of porphobilinogen each day, and about 5 grams of the pyrrole were isolated.
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The origin of the methyl group on C-1 of Ring A of the corrin ring of vitamin B(12) was investigated by (13)C magnetic resonance spectroscopy. The proton-decoupled (13)C spectra of vitamin B(12) synthesized from [5-(13)C]delta-aminolevulinic acid by Propionibacteria were obtained by Fourier-transform nuclear magnetic resonance of high resolution, and spectra of high-resolution proton magnetic resonance of the (13)C-labeled B(12) were also taken. The delta-carbon atom of delta-aminolevulinic acid is the source of seven or eight known positions of vitamin B(12), depending on whether the C-1 methyl group is also derived from the labeled substrate. We have found seven resonances whose chemical shifts enable us to identify the position of the (13)C atoms in the molecule from the assignment of Dodderell and Allerhand. We observed no (13)C resonance corresponding to the C-1 methyl group of Ring A. Furthermore, the proton magnetic resonance spectrum showed no spin-spin splitting of the proton peak at the tau values assigned for the H atoms in this methyl group. It would thus appear that the methyl group at C-1, which has been considered to have the same origin as the delta-methine bridge of porphyrins, does not originate from the delta-carbon atom of delta-aminolevulinic acid. This finding may aid in elucidation of the mechanism of synthesis of the functional isomer of vitamin B(12) and porphyrins. The (13)C-labeled vitamin B(12) enabled us to make further assignments to the (13)C magnetic resonance of the vitamin.