Insights into the Relationship between Cobamide Synthase and the Cell Membrane.
Autor: | Jeter VL; Department of Microbiology, University of Georgia, Athens, Georgia, USA., Escalante-Semerena JC; Department of Microbiology, University of Georgia, Athens, Georgia, USA jcescala@uga.edu. |
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Jazyk: | angličtina |
Zdroj: | MBio [mBio] 2021 Mar 23; Vol. 12 (2). Date of Electronic Publication: 2021 Mar 23. |
DOI: | 10.1128/mBio.00215-21 |
Abstrakt: | 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 (5' 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 (Copyright © 2021 Jeter and Escalante-Semerena.) |
Databáze: | MEDLINE |
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