Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Program in Chemical Biology, University of Michigan, Ann Arbor, MI, United States.
Department of Chemistry, University of Michigan, Ann Arbor, MI, United States.
Methods Enzymol. 2022;669:91-116. doi: 10.1016/bs.mie.2021.12.015. Epub 2022 Feb 3.
The cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster, SAM, and Cbl to carry out remarkable catalytic feats in a large number of biosynthetic pathways. However, despite the abundance of annotated Cbl-dependent radical SAM enzymes, relatively few molecular details exist regarding how these enzymes function. Traditionally, challenges associated with purifying and reconstituting Cbl-dependent radical SAM enzymes have hindered biochemical studies aimed at elucidating the structures and mechanisms of these enzymes. Herein, we describe a bottom-up approach that was used to crystallize OxsB, learn about the overall architecture of a Cbl-dependent radical SAM enzyme, and facilitate mechanistic studies. We report lessons learned from the crystallization of different states of OxsB, including the apo-, selenomethionine (SeMet)-labeled, and fully reconstituted form of OxsB that has a [4Fe-4S] cluster, SAM, and Cbl bound. Further, we suggest that, when appropriate, this bottom-up method can be used to facilitate studies on enzymes in this class for which there are challenges associated with purifying and reconstituting the active enzyme.
钴胺素 (Cbl)-依赖性自由基 S-腺苷甲硫氨酸 (SAM) 酶使用 [4Fe-4S] 簇、SAM 和 Cbl 在大量生物合成途径中完成显著的催化壮举。然而，尽管有大量注释的 Cbl-依赖性自由基 SAM 酶，但关于这些酶如何发挥作用的分子细节相对较少。传统上，与纯化和重组 Cbl-依赖性自由基 SAM 酶相关的挑战阻碍了旨在阐明这些酶的结构和机制的生化研究。在这里，我们描述了一种自下而上的方法，用于结晶 OxsB，了解 Cbl-依赖性自由基 SAM 酶的整体结构，并促进机制研究。我们报告了从 OxsB 的不同状态结晶中获得的经验教训，包括无辅因子、硒代蛋氨酸 (SeMet) 标记和完全重组的形式，其中含有 [4Fe-4S] 簇、SAM 和 Cbl 结合。此外，我们建议，在适当的情况下，这种自下而上的方法可用于促进对纯化和重组活性酶具有挑战性的此类酶的研究。
