Peptide Macrocyclization by a Bifunctional Endoprotease
Peptide Macrocyclization by a Bifunctional Endoprotease
复制标题
DOI：
10.1016/j.chembiol.2015.04.010
复制
发表时间：
2015-05-21
影响因子：
--
通讯作者：
Mylne, Joshua S.
中科院分区：
文献类型：
--
作者：
Bernath-Levin, Kalia;Nelson, Clark;Mylne, Joshua S.
蛋白水解酶通常会裂解多肽，但在某些条件下，它们可以将它们连接起来。向日葵种子含有14个残基的主干大环肽向日葵胰蛋白酶抑制因子1(SFTI-1)，其前体的成熟对天冬酰胺内肽酶(AEP)有遗传要求。为了提供更直接的证据，我们发展了一种原位分析方法，并使用O-18-水来证明SFTI-1从其线性前体中被切除并同时进行大环化。该反应在原位是低效的，但新发现的击穿途径可以通过在降解任何无环-SFTI之前将其内部二硫键还原为硫醇来掩盖这种低效。为了证实AEP可以直接进行切除/连接，我们在大肠杆菌中生产了几个重组植物AEP，其中一个来自菜豆的AEP既可以催化典型的切割反应，也可以催化切割依赖的分子内转肽来产生SFTI-1。我们认为，连接内切酶的进化使向日葵和菜豆等植物能够稳定生物活性多肽。
Proteases usually cleave peptides, but under some conditions, they can ligate them. Seeds of the common sunflower contain the 14-residue, backbone-macrocyclic peptide sunflower trypsin inhibitor 1 (SFTI-1) whose maturation from its precursor has a genetic requirement for asparaginyl endopeptidase (AEP). To provide more direct evidence, we developed an in situ assay and used O-18-water to demonstrate that SFTI-1 is excised and simultaneously macrocyclized from its linear precursor. The reaction is inefficient in situ, but a newfound breakdown pathway can mask this inefficiency by reducing the internal disulfide bridge of any acyclic-SFTI to thiols before degrading it. To confirm AEP can directly perform the excision/ligation, we produced several recombinant plant AEPs in E. coli, and one from jack bean could catalyze both a typical cleavage reaction and cleavage-dependent, intramolecular transpeptidation to create SFTI-1. We propose that the evolution of ligating endoproteases enables plants like sunflower and jack bean to stabilize bioactive peptides.