Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia.
Nat Commun. 2020 Mar 27;11(1):1575. doi: 10.1038/s41467-020-15418-2.
Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a k/K of 620 mM s, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.
天冬酰胺内肽酶（AEPs）在植物来源的环肽生物合成中催化关键的骨架环化步骤。在这里，我们报道了从苦瓜中鉴定出的两种 AEP，并对催化胰蛋白酶抑制剂环肽成熟的 MCoAEP2 进行了生化表征。重组生产的 MCoAEP2 催化线性环肽前体（MCoTI-II-NAL）的骨架环化，k/K 值为 620mM·s，是迄今为止报道的最快的环化酶之一。我们表明，MCoAEP2 可以在体外介导环肽前体的 N 端切除和 C 端环化。环化/水解的速率主要受 pH 值变化的影响，这可能在体内控制 AEP 介导的加工事件的顺序。此外，MCoAEP2 能够有效地催化具有抗血管生成活性的工程化 MCoTI-II 类似物的骨架环化。MCoAEP2 为通过直接化学方法以前无法获得的结构提供了增强的合成途径，并使胰蛋白酶抑制剂环肽在生物技术应用中的应用更加广泛。
