Journal
JOURNAL OF BACTERIOLOGY
Volume 182, Issue 17, Pages 4773-4782Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JB.182.17.4773-4782.2000
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The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Cop-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 mu mol) of B-12 derivatives was obtained as a crystalline sample. Analysis of the corrinoid sample of C. cochlearium by a combination of high-pressure liquid chromatography and W-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1. The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B-12(Co-beta-cyano-7 -adeninylcobamide) (60%) and factor A (Co-beta-cyano-7 - [2-methyl]adeninyl-cobamide) (20%). Authentic pseudovitamin B-12 was prepared by guided biosynthesis from cobinamide and adenine. Both pseudovitamin B-12 and its homologue, factor A, were subjected to complete spectroscopic analysis by W-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional H-1, C-13-, and N-15 nuclear magnetic resonance (NMR) spectroscopy. The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7 - [N-6-methyl]-adeninylcobamide, a previously unknown corrinoid. C. cochlearium thus biosynthesizes as its native complete B-12 cofactors the 7 -adeninyl-cobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.
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