Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Kalsec Inc., 3713W. Main St., Kalamazoo, MI 49006, USA.
Angew Chem Int Ed Engl. 2023 Apr 3;62(15):e202301370. doi: 10.1002/anie.202301370. Epub 2023 Feb 28.
Fe - and α-ketoglutarate-dependent halogenases and oxygenases can catalyze site-selective functionalization of C-H bonds via a variety of C-X bond forming reactions, but achieving high chemoselectivity for functionalization using non-native functional groups remains rare. The current study shows that directed evolution can be used to engineer variants of the dioxygenase SadX that address this challenge. Site-selective azidation of succinylated amino acids and a succinylated amine was achieved as a result of mutations throughout the SadX structure. The installed azide group was reduced to a primary amine, and the succinyl group required for azidation was enzymatically cleaved to provide the corresponding amine. These results provide a promising starting point for evolving additional SadX variants with activity on structurally distinct substrates and for enabling enzymatic C-H functionalization with other non-native functional groups.
铁和 α-酮戊二酸依赖的卤代酶和加氧酶可以通过各种 C-X 键形成反应催化 C-H 键的位点选择性功能化，但使用非天然官能团实现高化学选择性的功能化仍然很少见。本研究表明，定向进化可用于工程化双加氧酶 SadX 的变体，以解决这一挑战。通过 SadX 结构中的突变，实现了琥珀酰化氨基酸和琥珀酰化胺的选择性氮原子取代。引入的叠氮基团被还原为伯胺，而氮原子取代所需的琥珀酰基则通过酶促裂解被去除，从而提供相应的胺。这些结果为进一步进化具有不同结构底物活性的 SadX 变体以及用其他非天然官能团进行酶促 C-H 功能化提供了有希望的起点。
