Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5×102 to 1.8×104  m-1  s-1 . Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.

中文翻译：

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探索用于多功能蛋白功能化的细菌磷酸胆碱转移酶AnkX的底物范围。

特定于位点的蛋白质功能化已成为现代生命科学中必不可少的工具。在这里，基于标签的酶蛋白功能化技术是最通用的方法之一。然而，许多化学酶功能化策略受用于功能标记探针的酶的底物范围低的困扰。我们报告了细菌酶AnkX对衍生的CDP-胆碱类似物的广泛底物范围，并证明了AnkX催化的磷酸胆碱可用于具有多种不同功能的位点特异性一步和两步蛋白标记，显示快速二次传输速率为5×102到1.8×104 m-1 s-1。此外，我们还提出了一种针对感兴趣的蛋白质进行位点特异性双重标记的策略，基于AnkX的开发和Lem3酶的去标记功能。我们的结果为蛋白质功能化的广泛领域做出了贡献，为体外标记提供了一种有吸引力的基于化学酶标记的修饰策略。