PDF(Pubmed)
Abstract：
Radical S-adenosyl-L-methionine (rSAM) enzymes bind one or more Fe-S clusters and catalyze transformations that produce complex and structurally diverse natural products. One of the clusters, a 4Fe-4S cluster, binds and reductively cleaves SAM to generate the 5\'-deoxyadenosyl radical, which initiates the catalytic cycle by H-atom transfer from the substrate. The role(s) of the additional auxiliary Fe-S clusters (ACs) remains largely enigmatic. The rSAM enzyme PapB catalyzes the formation of thioether cross-links between the β-carbon of an Asp and a Cys thiolate found in the PapA peptide. One of the two ACs in the protein binds to the substrate thiol where, upon formation of a thioether bond, one reducing equivalent is returned to the protein. However, for the next catalytic cycle to occur, the protein must undergo an electronic state isomerization, returning the electron to the SAM-binding cluster. Using a series of iron-sulfur cluster deletion mutants, our data support a model whereby the isomerization is an obligatorily intermolecular electron transfer event that can be mediated by redox active proteins or small molecules, likely via the second AC in PapB. Surprisingly, a mixture of FMN and NADPH is sufficient to support both the reductive and the isomerization steps. These findings lead to a new paradigm involving intermolecular electron transfer steps in the activation of rSAM enzymes that require multiple iron-sulfur clusters for turnover. The implications of these results for the biological activation of rSAM enzymes are discussed.
摘要：
自由基S-腺苷-L-甲硫氨酸(rSAM)酶结合一个或多个Fe-S簇并催化转化,产生复杂且结构多样的天然产物。其中一个集群，4Fe-4S团簇，结合并还原裂解SAM以产生5'-脱氧腺苷自由基，它通过从底物转移H原子来启动催化循环。附加的辅助Fe-S团簇(AC)的作用在很大程度上仍然是神秘的。rSAM酶PapB催化在PapA肽中发现的Asp和Cys硫醇盐的β-碳之间形成硫醚交联。蛋白质中的两个AC之一与底物硫醇结合，在形成硫醚键时，一个还原当量返回到蛋白质。然而,为了下一个催化循环的发生，蛋白质必须经历电子态异构化，将电子返回到SAM结合簇。使用一系列铁硫簇缺失突变体，我们的数据支持一个模型，其中异构化是一个强制性的分子间电子转移事件，可以介导氧化还原活性蛋白或小分子，可能是通过PapB的第二个AC。令人惊讶的是,FMN和NADPH的混合物足以支持还原和异构化步骤。这些发现导致了一种新的范式，涉及rSAM酶活化中的分子间电子转移步骤，需要多个铁硫簇进行周转。讨论了这些结果对rSAM酶的生物活化的意义。
