Purification and characterization of sequential cobalamin-dependent radical SAM methylases ThnK and TokK in carbapenem β-lactam antibiotic biosynthesis.
Purification and characterization of sequential cobalamin-dependent radical SAM methylases ThnK and TokK in carbapenem β-lactam antibiotic biosynthesis.
复制标题
DOI：
10.1016/bs.mie.2021.09.014
复制
发表时间：
2022
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
ThnK和TokK是钴胺素依赖性自由基S-腺苷甲硫氨酸酶，催化常见碳青霉烯生物合成中间体的连续甲基化。ThnK是钴胺素依赖性自由基S-腺苷甲硫氨酸酶亚家族的早期特征成员。自从ThnK功能的首次发表以来，该领域取得了进展，我们在表达和纯化这种酶及其直系同源物TokK方面取得了方法学上的进步。一个优化的协议，以获得纯的和活性形式的酶，使我们能够表征他们的反应，并获得更深入的了解他们催化的顺序甲基化的动力学行为。我们在这里分享我们通过研究这些酶而开发的方法和策略。
ThnK and TokK are cobalamin-dependent radical S-adenosylmethionine enzymes that catalyze sequential methylations of a common carbapenem biosynthetic intermediate. ThnK was an early characterized member of the subfamily of cobalamin-dependent radical S-adenosylmethionine enzymes. Since initial publication of the ThnK function, the field has progressed, and we have made methodological strides in the expression and purification of this enzyme and its ortholog TokK. An optimized protocol for obtaining the enzymes in pure and active form has enabled us to characterize their reactions and gain greater insight into the kinetic behavior of the sequential methylations they catalyze. We share here the methods and strategy that we have developed through our study of these enzymes.