Numerous post-transcriptional modifications of transfer RNAs have vital roles in translation. The 2-methylthio-N⁶-isopentenyladenosine (ms²i⁶A) modification occurs at position 37 (A37) in transfer RNAs that contain adenine in position 36 of the anticodon, and serves to promote efficient A:U codon–anticodon base-pairing and to prevent unintended base pairing by near cognates, thus enhancing translational fidelity^1,2,3,4. The ms²i⁶A modification is installed onto isopentenyladenosine (i⁶A) by MiaB, a radical S-adenosylmethionine (SAM) methylthiotransferase. As a radical SAM protein, MiaB contains one [Fe₄S₄]_RS cluster used in the reductive cleavage of SAM to form a 5ʹ-deoxyadenosyl 5ʹ-radical, which is responsible for removing the C² hydrogen of the substrate⁵. MiaB also contains an auxiliary [Fe₄S₄]_aux cluster, which has been implicated^6,7,8,9 in sulfur transfer to C² of i⁶A37. How this transfer takes place is largely unknown. Here we present several structures of MiaB from Bacteroides uniformis. These structures are consistent with a two-step mechanism, in which one molecule of SAM is first used to methylate a bridging µ-sulfido ion of the auxiliary cluster. In the second step, a second SAM molecule is cleaved to a 5ʹ-deoxyadenosyl 5ʹ-radical, which abstracts the C² hydrogen of the substrate but only after C² has undergone rehybridization from sp² to sp³. This work advances our understanding of how enzymes functionalize inert C–H bonds with sulfur.

转移 RNA 的许多转录后修饰在翻译中起着至关重要的作用。2-甲硫基-N ⁶ -异戊烯基腺苷 (ms ² i ⁶ A) 修饰发生在转移 RNA 的第 37 位 (A37)，其在反密码子的第 36 位含有腺嘌呤，并用于促进有效的 A:U 密码子-反密码子碱基-配对并防止由近同源词引起的意外碱基配对，从而提高翻译保真度^1,2,3,4。ms ² i ⁶ A 修饰通过 MiaB（一种自由基S -腺苷甲硫氨酸 (SAM) 甲硫基转移酶）安装到异戊烯基腺苷 (i ^{6 A) 上。}作为一种自由基 SAM 蛋白，MiaB 含有一个 [Fe ₄S ₄ ] _RS簇用于SAM的还原裂解形成5'-脱氧腺苷5'-自由基，负责去除底物^{5的C 2}氢。MiaB 还包含一个辅助 [Fe ₄ S ₄ ]_辅助簇，它与硫转移到i ⁶ A37的 C ^{2中的6,7,8,9有关。}这种转移是如何发生的在很大程度上是未知的。在这里，我们展示了来自均匀拟杆菌的 MiaB 的几种结构. 这些结构与两步机制一致，其中一个 SAM 分子首先用于甲基化辅助簇的桥接 µ-硫化物离子。在第二步中，第二个 SAM 分子被切割成 5'-脱氧腺苷 5'-自由基，它提取底物的 C ²氢，但仅在 C ²经历从sp ²到sp ³的再杂化之后。这项工作促进了我们对酶如何使惰性 C-H 键与硫结合的理解。