TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases.
TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases.
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DOI：
10.1016/bs.mie.2017.07.007
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发表时间：
2017
影响因子：
--
通讯作者：
Booker SJ
中科院分区：
文献类型：
--
作者：
Blaszczyk AJ;Wang RX;Booker SJ
钴胺依赖的自由基S-腺苷甲硫氨酸甲基酶在许多抗生素、辅因子和其他重要天然产物的从头合成过程中起着至关重要的作用，但仍是自由基蛋氨酸酶中一个未被研究的亚类。除了由三个半胱氨酸残基连接的[4Fe-4S]簇外，这些酶还包含一个N末端的钴胺结合域。这些酶的体外研究一直受到严重限制，因为许多酶在大肠杆菌中过量生产时是不能溶解的或很少能溶解的。这种溶解性问题导致了许多小组要么从包涵体中提纯蛋白质，要么提纯通常缺乏适当辅因子掺入的可溶性蛋白质。在这里，我们使用TsrM作为模型来描述我们用来产生可溶性蛋白的方法，该蛋白以活性形式与钴胺和[4Fe-4S]簇辅因子结合而纯化。此外，我们强调了我们开发的方法，以表征纯化后的酶。
Cobalamin-dependent radical S-adenosylmethionine (SAM) methylases play vital roles in the de novo biosynthesis of many antibiotics, cofactors, and other important natural products, yet remain an understudied subclass of radical SAM (RS) enzymes. In addition to a [4Fe-4S] cluster that is ligated by three cysteine residues, these enzymes also contain an N-terminal cobalamin-binding domain. In vitro studies of these enzymes have been severely limited because many are insoluble or sparingly soluble upon their overproduction in E. coli. This solubility issue has led a number of groups either to purify the protein from inclusion bodies or to purify soluble protein that often lacks proper cofactor incorporation. Herein, we use TsrM as a model to describe methods that we have used to generate soluble protein that is purified in an active form with both cobalamin and [4Fe-4S] cluster cofactors bound. Additionally, we highlight the methods that we developed to characterize the enzyme following purification.
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