Insights into the Relationship between Cobamide Synthase and the Cell Membrane

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The late steps of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (59 phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis. IMPORTANCE Salmonella is a human pathogen of worldwide importance, and coenzyme B-12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B-12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B-12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B-12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.

Automated summary

This study presents the purification and biochemical characterization of the CobS enzyme, as well as its association with liposomes. Through in vitro and in vivo data, it expands our understanding of CobS and the significance of membrane-associated adenosylcobamide biosynthesis.