J Am Chem Soc. 2022 Aug 3;144(30):13556-13564. doi: 10.1021/jacs.2c03660. Epub 2022 Jul 22.
Piperazic acid (Piz) is a nonproteinogenic amino acid possessing a rare nitrogen-nitrogen bond. However, little is known about how Piz is incorporated into nonribosomal peptides, including whether adenylation domains specific to Piz exist. In this study, we show that free piperazic acid is directly adenylated and then incorporated into the incarnatapeptin nonribosomal peptides through isotopic incorporation studies. We also use reconstitution to demonstrate adenylation of free piperazic acid with a three-domain nonribosomal peptide synthetase from the incarnatapeptin gene cluster. We furthermore use bioinformatics and site-directed mutagenesis to outline consensus sequences for the adenylation of piperazic acid, which can now be used for the prediction of gene clusters linked to piperazic-acid-containing peptides. Finally, we discover a fusion protein of a piperazate synthase and an adenylation domain, highlighting the close biosynthetic relationship of piperazic acid formation and its adenylation. Altogether, our work demonstrates the evolution of biosynthetic systems for the activation of free piperazic acid through adenylation, a pathway we suggest is likely to be employed in the majority of pathways to piperazic-acid-containing peptides.
哌嗪酸（Piz）是一种非蛋白氨基酸，具有罕见的氮-氮键。然而，关于 Piz 如何被整合到非核糖体肽中，包括是否存在专门针对 Piz 的腺苷酸化结构域，人们知之甚少。在这项研究中，我们通过同位素掺入研究表明，游离哌嗪酸可直接被腺苷酸化，然后通过整合到 incarnatapeptin 非核糖体肽中。我们还使用重组来证明三结构域非核糖体肽合成酶对游离哌嗪酸的腺苷酸化。此外，我们使用生物信息学和定点突变来概述哌嗪酸腺苷酸化的共识序列，现在可以用于预测与含哌嗪酸肽相关的基因簇。最后，我们发现了哌嗪酸合酶和腺苷酸化结构域的融合蛋白，突出了哌嗪酸形成及其腺苷酸化的密切生物合成关系。总之，我们的工作证明了通过腺苷酸化激活游离哌嗪酸的生物合成系统的进化，我们认为该途径可能在大多数含哌嗪酸肽的途径中被采用。
