RNA methylation by Radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift
RNA methylation by Radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift
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DOI：
10.1073/pnas.1017781108
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发表时间：
2011-03-08
影响因子：
11.1
通讯作者：
Fujimori, Danica Galonic
中科院分区：
文献类型：
--
作者：
Yan, Feng;Fujimori, Danica Galonic
RlmN 和 Cfr 是自由基 SAM 酶，可修饰 23S 核糖体 RNA 中的单个腺苷核苷酸 -A2503。该核苷酸位于核糖体的肽基转移酶中心内，是许多抗生素的靶标。这些酶的一个不寻常的特征是它们能够对腺苷底物的脒碳进行甲基化。为了深入了解 RlmN 和 Cfr 催化甲基化的机制，进行了氘标记实验。这些实验表明，新引入的甲基是由 S-腺苷-L-甲硫氨酸 (SAM) 衍生的亚甲基片段和从底物脒碳迁移而来的氢原子组装而成。 5'-脱氧腺苷基自由基不是通过从脒碳上夺取氢原子来激活底物的腺苷核苷酸，而是从SAM的第二当量中夺取氢以形成SAM衍生的自由基阳离子。该物质或其相应的硫叶立德随后添加到底物中，引发氢化物转移和S-腺苷高半胱氨酸消除以完成甲基的形成。这些发现表明，RlmN 和 Cfr 不是甲基转移酶，而是甲基合酶。与之前描述的 5'-脱氧腺苷基和 3-氨基-3-羧丙基自由基一起，这些发现表明，连接到 SAM 中锍中心的所有三个碳原子都可以作为酶促反应中碳衍生自由基的前体。
RlmN and Cfr are Radical SAM enzymes that modify a single adenosine nucleotide-A2503-in 23S ribosomal RNA. This nucleotide is positioned within the peptidyl transferase center of the ribosome, which is a target of numerous antibiotics. An unusual feature of these enzymes is their ability to carry out methylation of amidine carbons of the adenosine substrate. To gain insight into the mechanism of methylation catalyzed by RlmN and Cfr, deuterium labeling experiments were carried out. These experiments demonstrate that the newly introduced methyl group is assembled from an S-adenosyl-L-methionine (SAM)-derived methylene fragment and a hydrogen atom that had migrated from the substrate amidine carbon. Rather than activating the adenosine nucleotide of the substrate by hydrogen atom abstraction from an amidine carbon, the 5'-deoxyadenosyl radical abstracts hydrogen from the second equivalent of SAM to form the SAM-derived radical cation. This species, or its corresponding sulfur ylide, subsequently adds into the substrate, initiating hydride shift and S-adenosylhomocysteine elimination to complete the formation of the methyl group. These findings indicate that rather than acting as methyl-transferases, RlmN and Cfr are methyl synthases. Together with the previously described 5'-deoxyadenosyl and 3-amino-3-carboxypropyl radicals, these findings demonstrate that all three carbon atoms attached to the sulfonium center in SAM can serve as precursors to carbon-derived radicals in enzymatic reactions.