Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
J Bacteriol. 2011 Oct;193(20):5623-8. doi: 10.1128/JB.05661-11. Epub 2011 Aug 5.
Salmonella enterica produces a proteinaceous microcompartment for B(12)-dependent 1,2-propanediol utilization (Pdu MCP). The Pdu MCP consists of catabolic enzymes encased within a protein shell, and its function is to sequester propionaldehyde, a toxic intermediate of 1,2-propanediol degradation. We report here that a short N-terminal region of the medium subunit (PduD) is required for packaging the coenzyme B(12)-dependent diol dehydratase (PduCDE) into the lumen of the Pdu MCP. Analysis of soluble cell extracts and purified MCPs by Western blotting showed that the PduD subunit mediated packaging of itself and other subunits of diol dehydratase (PduC and PduE) into the Pdu MCP. Deletion of 35 amino acids from the N terminus of PduD significantly impaired the packaging of PduCDE with minimal effects on its enzyme activity. Western blotting showed that fusing the 18 N-terminal amino acids of PduD to green fluorescent protein or glutathione S-transferase resulted in the association of these fusion proteins with the MCP. Immunoprecipitation tests indicated that the fusion proteins were encapsulated inside the MCP shell.
肠炎沙门氏菌产生一种蛋白质微隔间用于依赖 B(12)的 1,2-丙二醇利用 (Pdu MCP)。Pdu MCP 由包裹在蛋白质壳内的分解代谢酶组成，其功能是隔离丙醛，这是 1,2-丙二醇降解的一种有毒中间产物。我们在这里报告说，中等亚基 (PduD) 的短 N 端区域是将辅酶 B(12)依赖性二醇脱水酶 (PduCDE) 包装到 Pdu MCP 腔中的必需条件。通过 Western 印迹分析可溶性细胞提取物和纯化的 MCPs 表明，PduD 亚基介导自身和二醇脱水酶 (PduC 和 PduE) 的其他亚基包装到 Pdu MCP 中。从 PduD 的 N 端缺失 35 个氨基酸会严重损害 PduCDE 的包装，对其酶活性的影响最小。Western 印迹表明，将 PduD 的 18 个 N 端氨基酸融合到绿色荧光蛋白或谷胱甘肽 S-转移酶上会导致这些融合蛋白与 MCP 结合。免疫沉淀试验表明融合蛋白被包裹在 MCP 壳内。
