Joshi Sumedh, Fedoseyenko Dmytro, Mahanta Nilkamal, Begley Tadhg P
Department of Chemistry, Texas A&M University, College Station, TX, United States.
Department of Chemistry, Texas A&M University, College Station, TX, United States.
Methods Enzymol. 2018;606:179-198. doi: 10.1016/bs.mie.2018.05.002. Epub 2018 Jul 18.
Aminofutalosine synthase (MqnE) is a radical SAM enzyme involved in the futalosine-dependent menaquinone biosynthetic pathway. Its ability to add the 5'-deoxyadenosyl radical to the substrate-rather than abstract a hydrogen atom-and to catalyze radical addition to a stable benzene ring gives it a unique place in the radical SAM superfamily and required the development of new strategies for trapping radical intermediates. This chapter describes the methodologies used for enzyme overexpression, purification, and in vitro reconstitution. We also describe the development of fast, radical triggered, carbon-halogen bond fragmentation reactions for the trapping of intermediates. We anticipate that these methods will be of general use in the study of other transient enzymatic radicals.
氨基富他洛辛合酶（MqnE）是一种参与依赖富他洛辛的甲萘醌生物合成途径的自由基S-腺苷甲硫氨酸酶。它能够将5'-脱氧腺苷自由基添加到底物上，而不是夺取一个氢原子，并催化自由基加成到稳定的苯环上，这使其在自由基S-腺苷甲硫氨酸超家族中占据独特地位，也需要开发捕获自由基中间体的新策略。本章描述了用于酶的过表达、纯化和体外重组的方法。我们还描述了用于捕获中间体的快速、自由基引发的碳-卤键断裂反应的开发。我们预计这些方法将普遍用于研究其他瞬时酶自由基。
