Insights into the Relationship between Cobamide Synthase and the Cell Membrane.
Insights into the Relationship between Cobamide Synthase and the Cell Membrane.
复制标题
深入了解钴酰胺合酶与细胞膜之间的关系。
DOI：
10.1128/mbio.00215-21
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发表时间：
2021-03-23
期刊：
影响因子：
6.4
通讯作者：
Escalante-Semerena JC
中科院分区：
文献类型：
--
作者：
Jeter VL;Escalante-Semerena JC
沙门氏菌是世界范围内重要的人类病原体，辅酶B12对这种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用于分离钴酰胺合酶的方法，钴酰胺合酶是一种多位整合膜蛋白，催化辅酶B12生物合成的倒数第二步。钴酰胺是含钴的环状四吡咯，被生命的所有领域的细胞使用，但仅由一些细菌和古细菌从头产生。腺苷钴胺生物合成途径的“后期步骤”负责核苷酸环的组装，并且在从头合成和前体回收期间是必需的。这些步骤的特征在于咕啉环和较低配体碱的活化，活化的前体缩合为腺苷钴胺磷酸盐，并除去磷酸盐，产生完整的腺苷钴胺分子。激活的咕啉环和较低的配体碱基的缩合是由一个完整的膜蛋白，钴酰胺（5′磷酸）合酶（CobS），并代表了核苷酸环组装所需的两个途径的重要收敛。有趣的是，膜协会的倒数第二个步骤是保守的所有钴酰胺生产商，但这种协会的生理相关性是未知的。在这里，我们提出了纯化和生化特性的CobS酶的肠杆菌沙门氏菌肠亚种。肠血清型鼠伤寒菌株LT2，研究其与脂质体的关联，并量化脂质双层对其酶活性和底物亲和力的影响。我们报告的纯化方案，产生纯CobS蛋白，允许在体外功能分析。此外，我们报告的脂质体重建的CobS的方法，允许在磷脂双层的内膜蛋白的生理相关的研究。在体外和体内的数据报告，扩大我们的理解CobS和膜相关的腺苷钴胺生物合成的影响。
Salmonella is a human pathogen of worldwide importance, and coenzyme B12 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 B12 biosynthesis. 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.