The Cofactor of Tetrachloroethene Reductive Dehalogenase of Dehalospirillum multivorans Is Norpseudo‐B12, a New Type of a Natural Corrinoid

Abstract

The corrinoid cofactor of the tetrachloroethene reductive dehalogenase of Dehalospirillum multivorans was isolated in its Co β‐cyano form. This cofactor represents the main corrinoid found in D. multivorans cells. Analysis of the isolated cyano‐corrinoid by a combination of HPLC and UV/VIS‐absorbance spectroscopy revealed it to be nonidentical to a variety of known natural B 12 derivatives. From high‐resolution mass‐spectrometric analysis, the molecular formula of the corrinoid isolated from D. multivorans could be deduced as C 58 H 81 CoN 17 O 14 P. The sample of the novel corrinoid from D. multivorans was further analyzed by UV/VIS, CD, and one‐ and two‐dimensional 1 H‐, 13 C‐, and 15 N‐NMR spectroscopy, which indicated its structure to be closely related to that of pseudovitamin B 12 (Co β‐cyano‐7″‐adeninylcobamide). By the same means, the corrinoid could be shown to differ from pseudovitamin B 12 only by the lack of the methyl group attached to carbon 176, and, therefore, it was named norpseudovitamin B 12 (or, more precisely, 176‐norpseudovitamin B 12). Norpseudovitamin B 12 represents the first example of a ‘complete’ B 12‐cofactor that lacks one of the methyl groups of the cobamide moiety, indicating that the B 12‐biosynthetic pathway in D. multivorans differs from that of other organisms. X‐Ray crystal‐structures were determined for norpseudovitamin B 12 from D. multivorans and the analogues pseudovitamin B 12 and factor A (Co β‐cyano‐7″‐[2‐methyl]adeninylcobamide). These first accurate crystal structures of complete corrinoids with an adeninyl pseudonucleotide confirmed the expected coordination properties around Co and corroborated the close conformational similarity of the nucleotide moieties of norpseudovitamin B 12 and its two homologues.