Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes.
Biosynthesis of the sactipeptide Ruminococcin C by the human microbiome: Mechanistic insights into thioether bond formation by radical SAM enzymes.
复制标题
DOI：
10.1074/jbc.ra120.015371
复制
发表时间：
2020-12-04
期刊：
影响因子：
--
通讯作者：
Benjdia A
中科院分区：
文献类型：
--
作者：
Balty C;Guillot A;Fradale L;Brewee C;Lefranc B;Herrero C;Sandström C;Leprince J;Berteau O;Benjdia A
尽管它在人类健康中具有重要意义，但人类肠道微生物群的代谢潜力仍然知之甚少。Ruminococcin C（Ruminococcin C，RumC）是一种新型的核糖体合成和后修饰的肽，其生物合成需要两个自由基SAM酶（RumMC 1和RumMC 2）催化四个Cα-硫醚桥的形成。这些桥对于RumC对抗人类病原体（如产气荚膜梭菌）的抗生素特性是必不可少的，它们定义了两个发夹结构域，从而使这种sactipeptide（含硫至α-碳硫醚的肽）在天然产物中具有不寻常的结构。我们在这里报告RumMC 2的生化和光谱特征。EPR光谱和诱变数据支持RumMC 2是SPASM结构域自由基SAM酶大家族的成员，其特征在于存在三个[4Fe-4S]簇。我们还证明，这种酶启动其反应的Cα H-原子抽象，并能够催化非天然硫醚键的形成工程肽底物。出乎意料的是，我们的数据支持在Cα-硫醚桥LC-MS/MS裂解过程中形成酮亚胺而不是α，β-脱氢-氨基酸中间体。最后，我们探讨了前导肽和RiPP前体肽识别元件的作用，存在于无数的RiPP修饰酶中。总的来说，我们的数据支持一个更复杂的作用，为肽识别元件和核心肽的翻译后修饰的安装在RIPPs比以前预期的，并建议一个可能的反应中间体硫醚键的形成。
Despite its major importance in human health, the metabolic potential of the human gut microbiota is still poorly understood. We have recently shown that biosynthesis of Ruminococcin C (RumC), a novel ribosomally synthesized and posttranslationally modified peptide (RiPP) produced by the commensal bacterium Ruminococcus gnavus, requires two radical SAM enzymes (RumMC1 and RumMC2) catalyzing the formation of four Cα-thioether bridges. These bridges, which are essential for RumC's antibiotic properties against human pathogens such as Clostridium perfringens, define two hairpin domains giving this sactipeptide (sulfur-to-α-carbon thioether–containing peptide) an unusual architecture among natural products. We report here the biochemical and spectroscopic characterizations of RumMC2. EPR spectroscopy and mutagenesis data support that RumMC2 is a member of the large family of SPASM domain radical SAM enzymes characterized by the presence of three [4Fe-4S] clusters. We also demonstrate that this enzyme initiates its reaction by Cα H-atom abstraction and is able to catalyze the formation of nonnatural thioether bonds in engineered peptide substrates. Unexpectedly, our data support the formation of a ketoimine rather than an α,β-dehydro-amino acid intermediate during Cα-thioether bridge LC–MS/MS fragmentation. Finally, we explored the roles of the leader peptide and of the RiPP precursor peptide recognition element, present in myriad RiPP-modifying enzymes. Collectively, our data support a more complex role for the peptide recognition element and the core peptide for the installation of posttranslational modifications in RiPPs than previously anticipated and suggest a possible reaction intermediate for thioether bond formation.