Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase.
Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase.
复制标题
DOI：
10.1074/jbc.ra118.005369
复制
发表时间：
2018-11-09
期刊：
影响因子：
--
通讯作者：
Drennan CL
中科院分区：
文献类型：
--
作者：
Grell TAJ;Kincannon WM;Bruender NA;Blaesi EJ;Krebs C;Bandarian V;Drennan CL
Sactipeptides 是核糖体合成和翻译后修饰肽 (RiPP) 的一个亚类。它们在半胱氨酸残基的硫原子和受体残基的α-碳之间含有独特的硫醚键，称为硫氨酸键。这些连接是通过自由基化学形成的，对于 sactipeptides 的杀精、抗真菌和抗菌特性至关重要。形成这些连接的酶称为 sactisynthases，是 SPASM/Twitch 亚组中的 AdoMet 自由基酶，其结构尚未完全表征。在这里，我们展示了 SkfB 的 X 射线晶体结构和穆斯堡尔分析，SkfB 是来自枯草芽孢杆菌的一种糖合酶，参与产生芽孢杀灭因子 (SKF)。我们发现 SkfB 是一种模块化酶，其 N 端肽结合域包含 RiPP 识别元件 (RRE)、形成经典 AdoMet 自由基部分 (β/α)6 桶结构并显示 AdoMet 与 [4Fe-4S] 簇结合的中间域，以及所谓 Twitch 域特征的 C 端区域，其中包含辅助铁硫簇。值得注意的是，晶体学和穆斯堡尔分析都表明，SkfB 可以在辅助簇位点结合 [2Fe-2S] 簇，此前仅在另一种 AdoMet 自由基酶（吡咯喹啉醌生物合成酶 PqqE）结构中的 SPASM/Twitch 辅助簇位点中观察到过一次。总而言之，我们的研究结果表明，来自枯草芽孢杆菌的 SkfB 代表了一种独特的酶，包含在其他 AdoMet 自由基酶中观察到的几种结构特征。
Sactipeptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs). They contain a unique thioether bond, referred to as a sactionine linkage, between the sulfur atom of a cysteine residue and the α-carbon of an acceptor residue. These linkages are formed via radical chemistry and are essential for the spermicidal, antifungal, and antibacterial properties of sactipeptides. Enzymes that form these linkages, called sactisynthases, are AdoMet radical enzymes in the SPASM/Twitch subgroup whose structures are incompletely characterized. Here, we present the X-ray crystal structure to 1.29-Å resolution and Mössbauer analysis of SkfB, a sactisynthase from Bacillus subtilis involved in making sporulation killing factor (SKF). We found that SkfB is a modular enzyme with an N-terminal peptide-binding domain comprising a RiPP recognition element (RRE), a middle domain that forms a classic AdoMet radical partial (β/α)6 barrel structure and displays AdoMet bound to the [4Fe-4S] cluster, and a C-terminal region characteristic of the so-called Twitch domain housing an auxiliary iron-sulfur cluster. Notably, both crystallography and Mössbauer analyses suggest that SkfB can bind a [2Fe-2S] cluster at the auxiliary cluster site, which has been observed only once before in a SPASM/Twitch auxiliary cluster site in the structure of another AdoMet radical enzyme, the pyrroloquinoline quinone biosynthesis enzyme PqqE. Taken together, our findings indicate that SkfB from B. subtilis represents a unique enzyme containing several structural features observed in other AdoMet radical enzymes.