Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes
Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes
复制标题
DOI：
10.1038/nsmb.1597
复制
发表时间：
2009-06-01
影响因子：
16.8
通讯作者：
Weber-Ban, Eilika
中科院分区：
文献类型：
--
作者：
Striebel, Frank;Imkamp, Frank;Weber-Ban, Eilika
与真核生物中的泛素类似，细菌蛋白 Pup 附着在底物蛋白的赖氨酸残基上，从而靶向它们进行蛋白酶体降解。 It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here we have identified Dop (locus tag Rv2112) as the specific deamidase of Pup in Mycobacterium tuberculosis. Deamidation requires ATP as a cofactor but not its hydrolysis.此外，我们提供了PafA（基因座标签Rv2097）将脱酰胺化的Pup与蛋白酶体底物蛋白FabD和PanB连接的实验证据。异肽键的形成需要 ATP 水解为 ADP，这表明脱酰胺化的 Pup 通过其 C 端谷氨酸的磷酸化被激活以进行缀合。通过结合这些酶，我们在体外重建了完整的细菌泛素样修饰途径，包括由 Pup 脱酰胺酶 (Dop) 和 Pup 连接酶 (PafA) 催化的脱酰胺和连接步骤。
In analogy to ubiquitin in eukaryotes, the bacterial protein Pup is attached to lysine residues of substrate proteins, thereby targeting them for proteasomal degradation. It has been proposed that, before its attachment, Pup is modified by deamidation of its C-terminal glutamine to glutamate. Here we have identified Dop (locus tag Rv2112) as the specific deamidase of Pup in Mycobacterium tuberculosis. Deamidation requires ATP as a cofactor but not its hydrolysis. Furthermore, we provide experimental evidence that PafA (locus tag Rv2097) ligates deamidated Pup to the proteasomal substrate proteins FabD and PanB. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that deamidated Pup is activated for conjugation via phosphorylation of its C-terminal glutamate. By combining these enzymes, we have reconstituted the complete bacterial ubiquitin-like modification pathway in vitro, consisting of deamidation and ligation steps catalyzed by Pup deamidase (Dop) and Pup ligase (PafA).