d-amino acids endow peptides with diverse, desirable properties, but the post-translational and site-specific epimerization of l-amino acids into their d-counterparts is rare and chemically challenging. Bottromycins are ribosomally synthesized and post-translationally modified peptides that have overcome this challenge and feature a d-aspartate (d-Asp), which was proposed to arise spontaneously during biosynthesis. We have identified the highly unusual α/β-hydrolase (ABH) fold enzyme BotH as a peptide epimerase responsible for the post-translational epimerization of l-Asp to d-Asp during bottromycin biosynthesis. The biochemical characterization of BotH combined with the structures of BotH and the BotH–substrate complex allowed us to propose a mechanism for this reaction. Bioinformatic analyses of BotH homologs show that similar ABH enzymes are found in diverse biosynthetic gene clusters. This places BotH as the founding member of a group of atypical ABH enzymes that may be able to epimerize non-Asp stereocenters across different families of secondary metabolites.

d-氨基酸赋予肽多样化、理想的特性，但将l-氨基酸翻译后和位点特异性差向异构化为它们的d-对应物是罕见的并且在化学上具有挑战性。博曲霉素是核糖体合成和翻译后修饰的肽，克服了这一挑战并具有d-天冬氨酸 ( d - Asp)，建议在生物合成过程中自发产生。我们已经确定了非常不寻常的 α/β-水解酶 (ABH) 折叠酶 BotH 作为一种肽差向异构酶，负责将l - Asp翻译后差向异构化为d- 博曲霉素生物合成过程中的天冬氨酸。BotH 的生化特征与 BotH 和 BotH-底物复合物的结构相结合，使我们能够提出该反应的机制。对 BoTH 同源物的生物信息学分析表明，在不同的生物合成基因簇中发现了相似的 ABH 酶。这使 BotH 成为一组非典型 ABH 酶的创始成员，这些酶可能能够在不同的次级代谢产物家族中差向异构化非 Asp 立体中心。