Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, United States.
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States.
J Am Chem Soc. 2021 Aug 4;143(30):11690-11702. doi: 10.1021/jacs.1c05017. Epub 2021 Jul 20.
Lasso peptides are a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) defined by their threaded structure. Besides the class-defining isopeptide bond, other post-translational modifications (PTMs) that further tailor lasso peptides have been previously reported. Using genome mining tools, we identified a subset of lasso peptide biosynthetic gene clusters (BGCs) that are colocalized with genes encoding protein l-isoaspartyl methyltransferase (PIMT) homologues. PIMTs have an important role in protein repair, restoring isoaspartate residues formed from asparagine deamidation to aspartate. Here we report a new function for PIMT enzymes in the post-translational modification of lasso peptides. The PIMTs associated with lasso peptide BGCs first methylate an l-aspartate side chain found within the ring of the lasso peptide. The methyl ester is then converted into a stable aspartimide moiety, endowing the lasso peptide ring with rigidity relative to its unmodified counterpart. We describe the heterologous expression and structural characterization of two examples of aspartimide-modified lasso peptides from thermophilic Gram-positive bacteria. The lasso peptide cellulonodin-2 is encoded in the genome of actinobacterium , while lihuanodin is encoded in the genome of firmicute . Additional genome mining revealed PIMT-containing lasso peptide BGCs in 48 organisms. In addition to heterologous expression, we have reconstituted PIMT-mediated aspartimide formation in vitro, showing that lasso peptide-associated PIMTs transfer methyl groups very rapidly as compared to canonical PIMTs. Furthermore, in stark contrast to other characterized lasso peptide PTMs, the methyltransferase functions only on lassoed substrates.
套索肽是一类核糖体合成和翻译后修饰的肽（RiPPs），其特征为螺旋结构。除了定义类别的异肽键外，以前还报道了其他翻译后修饰（PTM）进一步修饰套索肽。使用基因组挖掘工具，我们鉴定了一组套索肽生物合成基因簇（BGC），这些 BGC 与编码蛋白质 l-异天冬氨酸甲基转移酶（PIMT）同源物的基因共定位。PIMTs 在蛋白质修复中起着重要作用，将由天冬酰胺脱酰胺形成的异天冬氨酸残基恢复为天冬氨酸。在这里，我们报告了 PIMT 酶在套索肽翻译后修饰中的新功能。与套索肽 BGC 相关的 PIMTs 首先甲基化套索肽环内的 l-天冬氨酸侧链。然后，甲酯转化为稳定的天冬酰胺亚胺部分，使套索肽环相对于其未修饰的对应物具有刚性。我们描述了两种来自嗜热革兰氏阳性细菌的天冬酰胺亚胺修饰套索肽的异源表达和结构特征。放线菌基因组中编码了套索肽 cellulonodin-2，而硬壁菌基因组中编码了 lihuanodin。额外的基因组挖掘揭示了在 48 个生物体中含有 PIMT 的套索肽 BGC。除了异源表达外，我们还在体外重建了 PIMT 介导的天冬酰胺亚胺形成，结果表明与典型的 PIMT 相比，套索肽相关的 PIMT 非常迅速地转移甲基。此外，与其他已鉴定的套索肽 PTM 形成鲜明对比的是，甲基转移酶仅在套索化底物上起作用。
