Chemical Biology Center (KBC), Department of Chemistry, Umeå University, Linnaeus väg 10, 90187, Umeå, Sweden.
Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistrasse 52, 20246, Hamburg, Germany.
Chembiochem. 2019 Sep 16;20(18):2336-2340. doi: 10.1002/cbic.201900200. Epub 2019 Aug 21.
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 5×10 to 1.8×10 m s . 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.
基于标签的酶法蛋白质功能化技术是现代生命科学中不可或缺的工具。然而，许多化学酶法功能化策略都受到所使用酶的功能化探针的底物范围的限制。我们报道了细菌酶 AnkX 对衍生化 CDP-胆碱类似物的广泛底物范围，并证明 AnkX 催化的磷酰化可以用于具有广泛不同功能的蛋白质的一步和两步的定点标记，显示出快速的二级转移速率为 5×10 到 1.8×10 m s 。此外，我们还提出了一种基于 AnkX 的定点双标记策略和酶 Lem3 的去标签功能，用于对感兴趣的蛋白质进行定点双标记。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签的修饰策略。
