Kincannon William M, Bruender Nathan A, Bandarian Vahe
Department of Chemistry , University of Utah , 315 South 1400 East , Salt Lake City , Utah 84112 , United States.
Biochemistry. 2018 Aug 14;57(32):4816-4823. doi: 10.1021/acs.biochem.8b00537. Epub 2018 Jul 24.
Sporulation killing factor (SKF) is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by Bacillus. SKF contains a thioether cross-link between the α-carbon at position 40 and the thiol of Cys32, introduced by a member of the radical S-adenosyl-l-methionine (SAM) superfamily, SkfB. Radical SAM enzymes employ a 4Fe-4S cluster to bind and reductively cleave SAM to generate a 5'-deoxyadenosyl radical. SkfB utilizes this radical intermediate to abstract the α-H atom at Met40 to initiate cross-linking. In addition to the cluster that binds SAM, SkfB also has an auxiliary cluster, the function of which is not known. We demonstrate that a substrate analogue with a cyclopropylglycine (CPG) moiety replacing the wild-type Met40 side chain forgoes thioether cross-linking for an alternative radical ring opening of the CPG side chain. The ring opening reaction also takes place with a catalytically inactive SkfB variant in which the auxiliary Fe-S cluster is absent. Therefore, the CPG-containing peptide uncouples H atom abstraction from thioether bond formation, limiting the role of the auxiliary cluster to promoting thioether cross-link formation. CPG proves to be a valuable tool for uncoupling H atom abstraction from peptide modification in RiPP maturases and demonstrates potential to leverage RS enzyme reactivity to create noncanonical amino acids.
芽孢形成杀伤因子（SKF）是一种由芽孢杆菌产生的核糖体合成及翻译后修饰肽（RiPP）。SKF在第40位的α-碳与Cys32的硫醇之间含有一个硫醚交联键，该交联键由自由基S-腺苷-L-甲硫氨酸（SAM）超家族成员SkfB引入。自由基SAM酶利用一个4Fe-4S簇结合并还原裂解SAM以生成一个5'-脱氧腺苷自由基。SkfB利用这个自由基中间体夺取Met40上的α-H原子以启动交联反应。除了结合SAM的簇之外，SkfB还具有一个辅助簇，其功能尚不清楚。我们证明，一种用环丙基甘氨酸（CPG）部分取代野生型Met40侧链的底物类似物放弃了硫醚交联反应，转而进行CPG侧链的另一种自由基开环反应。开环反应也发生在一种缺乏辅助Fe-S簇的无催化活性的SkfB变体中。因此，含CPG的肽将氢原子夺取与硫醚键形成解偶联，将辅助簇的作用限制为促进硫醚交联形成。CPG被证明是一种用于使RiPP成熟酶中的氢原子夺取与肽修饰解偶联的有价值工具，并展示了利用RS酶反应性创造非天然氨基酸的潜力。
