Native corrinoids from Clostridium cochlearium are adeninylcobamides: spectroscopic analysis and identification of pseudovitamin B(12) and factor A

Abstract

The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives. From 50 g of frozen cells, approximately 2 mg (1.5 micromol) 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 UV-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]adeninylcobamide) (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 UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N 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"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.

FIG. 1

Structural formulae of vitamin B₁₂ derivatives. The general formulae are given on the left; variation of the organic ligand R (Me, methyl; Ado, 5′-deoxy-5′-adenosyl) and/or of the nucleotide base give rise to the different cobamides. (Top) Base-on cobamides vitamin B₁₂ (cyanocobalamin), methylcobalamin, coenzyme B₁₂ (5′-deoxy-5′-adenosylcobalamin), pseudovitamin B₁₂ (Co_β-cyano-7"-adeninylcobamide), Co_β-methyl-7"-adeninylcobamide, factor A (Co_β-cyano-7"-[2-methyl]adeninylcobamide), Co_β-methyl-7"-[2-methyl]adeninylcobamide, and Co_β-cyano-7"-[N⁶-methyl]adeninylcobamide. (Bottom) Base-off cobamides pseudocoenzyme B₁₂ (5′-deoxy-5′-adenosyl-7"-adeninylcobamide) and adenosyl factor A (5′-deoxy-5′-adenosyl-7"-[2-methyl]-adeninylcobamide).

FIG. 2

Atom numbering of pseudovitamin B₁₂ (and factor A) used for the description of NMR results (44). The atom numbering for the DMB of vitamin B₁₂ is also shown.

FIG. 3

(Top) HPLC chromatogram of the corrinoid sample isolated from C. cochlearium. (Bottom) UV-Vis spectra of the new corrinoid (fraction II of the HPLC chromatogram) (see Materials and Methods for technical details).

FIG. 4

The 500-MHz ¹H NMR spectra of aqueous solutions of pseudovitamin B₁₂, factor A, and the corrinoid sample isolated from C. cochlearium are shown. Two signals, tentatively assigned to 7"-[N⁶-methyl]adeninylcobamide, are labeled with a question mark.