Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Microbiology, University of Georgia, Athens, Georgia, USA
mBio. 2021 Mar 23;12(2):e00215-21. doi: 10.1128/mBio.00215-21.
Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced 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 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 subsp. 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 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. and data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis. is a human pathogen of worldwide importance, and coenzyme B 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 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during coenzyme B 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-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.
钴胺素是一种含钴的环状四吡咯，存在于所有生命领域的细胞中，但仅由某些细菌和古菌产生。腺苷钴胺素生物合成途径的“后期步骤”负责核苷酸环的组装，并且在合成和前体回收过程中是必需的。这些步骤的特征是钴胺素环和较低配体碱基的激活、活化前体与腺苷钴胺素磷酸的缩合，以及磷酸的去除，从而生成完整的腺苷钴胺素分子。激活的钴胺素环和较低配体碱基的缩合由一个完整的膜蛋白——钴胺素（5' 磷酸）合酶（CobS）完成，这代表了核苷酸环组装所必需的两条途径的重要收敛。有趣的是，这种前体步骤的膜结合在所有钴胺素产生菌中都是保守的，但这种结合的生理相关性尚不清楚。在这里，我们展示了肠杆菌亚种。LT2 菌株的 CobS 酶的纯化和生化特性，研究了它与脂质体的关联，并量化了脂质双层对其酶活性和底物亲和力的影响。我们报告了一种纯化方案，可产生纯 CobS 蛋白，从而允许进行功能分析。此外，我们还报告了一种用于 CobS 脂质体重建的方法，允许在磷脂双层中进行这种内膜蛋白的生理相关研究。这里报道的数据扩展了我们对 CobS 的理解，以及膜相关腺苷钴胺素生物合成的意义。是一种具有全球重要性的人类病原体，辅酶 B 对该细菌的致病生活方式至关重要。这里报道的工作的重要性在于改进了分离钴胺素合酶的方法，钴胺素合酶是催化辅酶 B 生物合成中最后一步的多结构域整合膜蛋白。这一进展是分析负责核苷酸环组装的多酶复合物的重要一步，也是分析从环境中吸收不完全钴胺素的重要一步。我们提出，迄今为止测序的每一种产生辅酶 B 的细菌和古菌都可能将钴胺素合酶定位于细胞膜上。关于钴胺素合酶在脂质双层背景下的功能的新知识，推进了我们对该酶的理解，并为上述多酶复合物的功能结构分析奠定了基础。
