Identification of purinyl-cobamide as a novel corrinoid cofactor of tetrachloroethene reductive dehalogenases in Desulfitobacterium spp.

Author

Meng Bi, University of Tennessee - KnoxvilleFollow

Date of Award

12-2015

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Frank E. Loeffler

Committee Members

Alison Buchan, Karen lloyd, Qiang He

Abstract

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.

Recommended Citation

Bi, Meng, "Identification of purinyl-cobamide as a novel corrinoid cofactor of tetrachloroethene reductive dehalogenases in Desulfitobacterium spp.. " Master's Thesis, University of Tennessee, 2015.
https://trace.tennessee.edu/utk_gradthes/3561