Identification of purinyl-cobamide as a novel corrinoid cofactor of tetrachloroethene reductive dehalogenases in \u3ci\u3eDesulfitobacterium\u3c/i\u3e spp.

Corrinoids (e.g. vitamin B12) [cyanocobalamin] are a group of structurally similar, cobaltcontaining tetrapyrrole compounds involved in a number of important biochemical reactions. In organohalide respiration, vitamin B12 analogues carrying different lower bases are obligate cofactors for the reductive dehalogenases (RDases) that catalyze reductive dechlorination reactions. The focus of this research was on the isolation and characterization of a novel natural corrinoid cofactor that enables the dechlorination-coupled energy conservation in organohaliderespiring Desulfitobacterium strains. Analysis of the purified corrinoid in the cyano form using a combination of high-performance liquid chromatography (HPLC), UV-Vis [Ultraviolet–visible spectroscopy] and ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS) demonstrated that this novel corrinoid is different than the currently known naturally occurring corrinoids in the lower base structure. Analysis by 15N [nitrogen isotope 15] isotope labeling methods suggested a lower base with the molecular formula C5H3N4 [purine] (ligand form). Proton (1H) and correlation spectroscopy (COSY) NMR [Nuclear magnetic resonance] experiments corroborated that purine is the lower base, and Coα-purinyl-Coβ-cyanocobamide [cobalt alpha-purinyl-cobalt beta-cyanocobamide] (purinylcobamide) is a novel corrinoid. The dechlorination-supporting function of purinylcobamide was validated using corrinoid-auxotrophic Dehalobacter restrictus (Dhb) and Dehalococcoides mccartyi (Dhc) pure cultures expressing distinct RDases. Indistinguishable bacterial growth and dechlorination rates compared to vitamin B12-amended cultures were observed in Dhb but not Dhc cultures, demonstrating iv distinct cofactor requirements. Overall, the discovery of purinylcobamide emphasizes that the diversity and functions of corrinoids, especially the lower base structures to organaohalide-respiring bacteria, need to be fully understood to implement successful bioremediation for chlorinated solvents contamination