Radical S‐adenosyl methionine enzymes catalyze a diverse repertoire of post‐translational modifications in protein and peptide substrates. Among these, an exceptional and mechanistically obscure example is the installation of α‐keto‐β‐amino acid residues by formal excision of a tyrosine‐derived tyramine unit. The responsible spliceases are key maturases in a widespread family of natural products termed spliceotides that comprise potent protease inhibitors, with the installed β‐residues being crucial for bioactivity. Here, we established the in vitro activity of the model splicease PcpXY to interrogate the mechanism of non‐canonical protein splicing. Identification of shunt and coproducts, deuterium labeling studies, and density functional theory energy calculations of hypothesized intermediates support a mechanism involving hydrogen abstraction at tyrosine Cα as the initial site of peptide radical formation and release of 4‐hydroxybenzaldehyde as the tyrosine‐derived coproduct. The data illuminate key features of this unprecedented radical‐mediated biotransformation yielding ketoamide pharmacophores that are also present in peptidomimetic therapeutics.

中文翻译：

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自由基 S-腺苷甲硫氨酸肽剪接酶形成翻译后 α-酮-β-氨基酸的机制见解


自由基 S-腺苷甲硫氨酸酶催化蛋白质和肽底物中的多种翻译后修饰。其中，一个特殊且机制上晦涩难懂的例子是通过正式切除酪氨酸衍生的酪胺单元来安装 α-酮-β-氨基酸残基。负责的剪接酶是广泛的天然产物家族（称为剪接肽）中的关键成熟酶，由有效的蛋白酶抑制剂组成，安装的 β 残基对生物活性至关重要。在这里，我们建立了模型剪接酶 PcpXY 的体外活性，以询问非经典蛋白剪接的机制。分流和共产物的鉴定、氘标记研究以及假设中间体的密度泛函理论能量计算支持一种机制，即酪氨酸 Cα 处的氢提取作为肽自由基形成的初始位点，并释放 4-羟基苯甲醛作为酪氨酸衍生的副产物。这些数据阐明了这种前所未有的自由基介导的生物转化的关键特征，产生的酮酰胺药效团也存在于拟肽治疗剂中。