Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Nat Chem. 2022 Nov;14(11):1325-1334. doi: 10.1038/s41557-022-01022-y. Epub 2022 Aug 18.
Microviridins and other ω-ester-linked peptides, collectively known as graspetides, are characterized by side-chain-side-chain linkages installed by ATP-grasp enzymes. Here we report the discovery of a family of graspetides, the gene clusters of which also encode an O-methyltransferase with homology to the protein repair catalyst protein L-isoaspartyl methyltransferase. Using heterologous expression, we produced fuscimiditide, a ribosomally synthesized and post-translationally modified peptide (RiPP). NMR analysis of fuscimiditide revealed that the peptide contains two ester cross-links forming a stem-loop macrocycle. Furthermore, an unusually stable aspartimide moiety is found within the loop macrocycle. We fully reconstituted fuscimiditide biosynthesis in vitro including formation of the ester and aspartimide moieties. The aspartimide moiety embedded in fuscimiditide hydrolyses regioselectively to isoaspartate. Surprisingly, this isoaspartate-containing peptide is also a substrate for the L-isoaspartyl methyltransferase homologue, thus driving any hydrolysis products back to the aspartimide form. Whereas an aspartimide is often considered a nuisance product in protein formulations, our data suggest that some RiPPs have aspartimide residues intentionally installed via enzymatic activity.
微病毒素和其他 ω-酯键连接的肽，统称为graspetides，其特征是通过 ATP-抓握酶安装侧链-侧链连接。在这里，我们报告了一类 graspetides 的发现，其基因簇还编码一种与蛋白质修复催化剂蛋白 L-异天冬氨酸甲基转移酶同源的 O-甲基转移酶。我们通过异源表达生产了 fuscimiditide，一种核糖体合成和翻译后修饰的肽（RiPP）。对 fuscimiditide 的 NMR 分析表明，该肽含有两个形成茎环大环的酯键交联。此外，在环大环中发现了一个异常稳定的天冬酰胺亚基。我们在体外完全重建了 fuscimiditide 的生物合成，包括酯和天冬酰胺亚基的形成。嵌入 fuscimiditide 中的天冬酰胺亚基具有区域选择性水解为异天冬氨酸。令人惊讶的是，这种含有异天冬氨酸的肽也是 L-异天冬氨酸甲基转移酶同源物的底物，从而将任何水解产物转回天冬酰胺亚基形式。虽然天冬酰胺亚基通常被认为是蛋白质制剂中的一种有害产物，但我们的数据表明，一些 RiPPs 是通过酶活性有意地安装了天冬酰胺亚基。
