Designer installation of a substrate recruitment domain to tailor enzyme specificity.
Designer installation of a substrate recruitment domain to tailor enzyme specificity.
复制标题
设计师安装底物招募域来定制酶特异性。
DOI：
10.1038/s41589-022-01206-0
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发表时间：
2023
影响因子：
14.8
通讯作者：
Kuhlman,Brian
中科院分区：
文献类型：
--
作者：
Park,Rodney;Ongpipattanakul,Chayanid;Nair,SatishK;Bowers,AlbertA;Kuhlman,Brian
修饰肽和蛋白质的混杂酶是标记生物分子的有力工具；然而，将这些修饰导向所需的底物可能具有挑战性。在这里，我们使用计算界面设计在混杂酶儿茶酚甲基转移酶的活性位点附近安装一个底物识别域。这种设计方法有效地将底物识别与催化位点分离，并促进了被招募域识别的肽的修饰。我们确定了这种新型多结构域酶SH3-588的晶体结构，这表明它与我们的设计非常吻合。SH3-588甲基化定向肽的催化效率超过野生型酶的1000倍以上，而缺乏定向识别序列的肽没有显示出更高的效率。在竞争实验中，设计酶优先修饰定向底物而非定向底物，这表明我们可以使用设计的招募结构域在复杂的多底物环境中指导靶蛋白上特定序列基序的翻译后修饰。
Promiscuous enzymes that modify peptides and proteins are powerful tools for labeling biomolecules; however, directing these modifications to desired substrates can be challenging. Here, we use computational interface design to install a substrate recognition domain adjacent to the active site of a promiscuous enzyme, catecholO-methyltransferase. This design approach effectively decouples substrate recognition from the site of catalysis and promotes modification of peptides recognized by the recruitment domain. We determined the crystal structure of this novel multidomain enzyme, SH3-588, which shows that it closely matches our design. SH3-588 methylates directed peptides with catalytic efficiencies exceeding the wild-type enzyme by over 1,000-fold, whereas peptides lacking the directing recognition sequence do not display enhanced efficiencies. In competition experiments, the designer enzyme preferentially modifies directed substrates over undirected substrates, suggesting that we can use designed recruitment domains to direct post-translational modifications to specific sequence motifs on target proteins in complex multisubstrate environments.