Department of Chemistry, Johns Hopkins University, Baltimore, MD, United States.
Department of Chemistry, Johns Hopkins University, Baltimore, MD, United States.
Methods Enzymol. 2022;669:29-44. doi: 10.1016/bs.mie.2021.09.014. Epub 2021 Oct 26.
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.
ThnK 和 TokK 是钴胺素依赖的自由基 S-腺苷甲硫氨酸酶，可催化共同碳青霉烯生物合成中间体的顺序甲基化。ThnK 是钴胺素依赖的自由基 S-腺苷甲硫氨酸酶亚家族的早期特征成员。自 ThnK 功能的最初发表以来，该领域已经取得了进展，我们在该酶及其同源物 TokK 的表达和纯化方面取得了方法上的进步。获得酶的纯品和活性形式的优化方案使我们能够对其反应进行表征，并深入了解它们催化的顺序甲基化的动力学行为。我们在这里分享通过对这些酶的研究而开发的方法和策略。
