小柯机器人

近日，美国宾夕法尼亚州立大学Squire J. Booker等研究人员合作揭示自由基SAM酶MiaB对tRNA甲硫醇化作用的结构基础。这一研究成果于2021年9月15日在线发表在国际学术期刊《自然》上。

研究人员表示，转移RNA的许多转录后修饰在翻译中具有重要作用。2-甲硫基-N6-异戊烯基腺苷（ms²i⁶A）修饰发生在反密码子36位含有腺嘌呤的转移RNA的37位（A37），用于促进有效的A:U密码子-反密码子碱基配对，并防止近亲的非预期碱基配对，从而提高翻译的保真度。ms²i⁶A修饰由MiaB添加到异戊烯基腺苷（i⁶A）上，MiaB是一种自由基的S-腺苷蛋氨酸（SAM）甲基转移酶。作为一个自由基SAM蛋白，MiaB含有一个[Fe₄S₄]_RS簇，用于SAM的还原性裂解，形成一个5-脱氧腺苷5-自由基，负责去除底物的C²氢。MiaB还含有一个辅助的[Fe₄S₄]_aux簇，它被认为参与了硫向i⁶A37的C²转移。这种转移是如何发生的在很大程度上是未知的。

研究人员报道了单形拟杆菌MiaB的几个结构。这些结构与一个两步机制相一致，其中一个SAM分子首先被用来甲基化辅助团簇的桥接µ-硫代离子。在第二步中，第二个SAM分子被裂解为一个5-脱氧腺苷5-自由基，它抽取了底物的C²氢，但只在C²经历了从sp²到sp³的重新杂交之后。这项工作推进了人们对酶如何用硫使惰性C-H键功能化的理解。

附：英文原文

Title: Structural basis for tRNA methylthiolation by the radical SAM enzyme MiaB

Author: Esakova, Olga A., Grove, Tyler L., Yennawar, Neela H., Arcinas, Arthur J., Wang, Bo, Krebs, Carsten, Almo, Steven C., Booker, Squire J.

Issue&Volume: 2021-09-15

Abstract: 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 fidelity1,2,3,4. 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 5-deoxyadenosyl 5-radical, which is responsible for removing the C2 hydrogen of the substrate5. MiaB also contains an auxiliary [Fe4S4]aux cluster, which has been implicated6,7,8,9 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 5-deoxyadenosyl 5-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.

DOI: 10.1038/s41586-021-03904-6

Source: https://www.nature.com/articles/s41586-021-03904-6

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