Radical S-adenosyl-l-methionine (RS) enzymes operate on a variety of substrates and catalyze a wide range of complex radical-mediated transformations. Radical non-α-carbon thioether peptides (ranthipeptides) are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs). The RS enzyme PapB catalyzes the formation of thioether cross-links between Cys/Asp (or Cys/Glu) residues located in six Cys-X₃-Asp/Glu motifs. In this report, using a minimal substrate that contains a single cross-link motif, we explore the substrate scope of the PapB and show that the enzyme is highly promiscuous and will accept a variety of Cys-X_n-Asp sequences where n = 0–6. Moreover, we show that the enzyme will introduce in-line and nested thioether cross-links independently in peptide sequences that contain two motifs derived from the wild-type sequence. Additionally, the enzyme accepts peptides that contain d-amino acids at either the Cys or the Asp position. These observations are leveraged to produce a thioether cyclized analogue of the FDA-approved therapeutic agent octreotide, with a Cys-Glu cross-link replacing the disulfide that is found in the drug. These findings highlight the remarkable substrate tolerance of PapB and show the utility of RS RiPP maturases in biotechnological applications.

中文翻译：

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利用自由基 S-腺苷-L-甲硫氨酸 RiPP 成熟酶的底物混杂性进行分子内肽交联应用

自由基S-腺苷-L-甲硫氨酸 (RS) 酶作用于多种底物并催化范围广泛的复杂自由基介导的转化。自由基非α-碳硫醚肽（ranthipeptides）是一类核糖体合成和翻译后修饰的肽（RiPPs）。RS酶PapB催化位于六个Cys-X ₃ -Asp/Glu基序中的Cys/Asp（或Cys/Glu）残基之间形成硫醚交联。在本报告中，使用包含单个交联基序的最小底物，我们探索了 PapB 的底物范围，并表明该酶是高度混杂的，将接受各种 Cys-X _n -Asp 序列，其中n= 0–6。此外，我们表明该酶将在包含两个源自野生型序列的基序的肽序列中独立地引入内嵌和嵌套的硫醚交联。此外，该酶接受在 Cys 或 Asp 位置含有d-氨基酸的肽。利用这些观察结果生产 FDA 批准的治疗剂奥曲肽的硫醚环化类似物，其中 Cys-Glu 交联取代了药物中发现的二硫化物。这些发现突出了 PapB 显着的底物耐受性，并显示了 RS RiPP 成熟酶在生物技术应用中的实用性。