Simon J. de Veer, David J. Craik, Fabian B. H. Rehm
{"title":"Highly Efficient Transpeptidase-Catalyzed Isopeptide Ligation","authors":"Simon J. de Veer, David J. Craik, Fabian B. H. Rehm","doi":"10.1021/jacs.4c11964","DOIUrl":null,"url":null,"abstract":"Transpeptidases are specialized enzymes that have evolved for site-selective modification of peptides and proteins at their backbone termini. Approaches for adapting transpeptidases to catalyze side chain modifications are substantially more restricted, and typically rely on large recognition tags or require specific reaction conditions that are not easily compatible with broader applications. Here we show that the engineered asparaginyl ligase <i>Oa</i>AEP1 catalyzes direct isopeptide ligation by accepting an internal 2,3-diaminopropionic acid (Dap) residue adjacent to Leu, a motif that mimics the canonical N-terminal Gly-Leu substrate. These reactions proceed efficiently at near-neutral pH without any required additives, enabling straightforward formation of diverse isopeptide-linked products under simple reaction conditions. We demonstrate that <i>Oa</i>AEP1-catalyzed isopeptide ligation can be utilized for site-selective side chain labeling at an introduced Dap residue with minimal off-target labeling of Lys residues. Additionally, we generate engineered peptide topologies via intramolecular side chain-to-tail cross-links and produce direct protein–cyclic peptide fusions via efficient intermolecular ligation. We also show that <i>Oa</i>AEP1-catalyzed isopeptide ligation extends to <span>d</span>-peptide acceptors containing a retro-inverso <span>d</span>-Leu-<span>d</span>-Dap motif. This capability further expands the range and complexity of isopeptide-linked products that can be accessed with <i>Oa</i>AEP1, which we exemplify by forming a hybrid <span>d</span>-/<span>l</span>- bicyclic peptide topology where both termini are linked to internal side chains.","PeriodicalId":49,"journal":{"name":"Journal of the American Chemical Society","volume":"13 1","pages":""},"PeriodicalIF":14.4000,"publicationDate":"2024-12-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of the American Chemical Society","FirstCategoryId":"92","ListUrlMain":"https://doi.org/10.1021/jacs.4c11964","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MULTIDISCIPLINARY","Score":null,"Total":0}
引用次数: 0
Abstract
Transpeptidases are specialized enzymes that have evolved for site-selective modification of peptides and proteins at their backbone termini. Approaches for adapting transpeptidases to catalyze side chain modifications are substantially more restricted, and typically rely on large recognition tags or require specific reaction conditions that are not easily compatible with broader applications. Here we show that the engineered asparaginyl ligase OaAEP1 catalyzes direct isopeptide ligation by accepting an internal 2,3-diaminopropionic acid (Dap) residue adjacent to Leu, a motif that mimics the canonical N-terminal Gly-Leu substrate. These reactions proceed efficiently at near-neutral pH without any required additives, enabling straightforward formation of diverse isopeptide-linked products under simple reaction conditions. We demonstrate that OaAEP1-catalyzed isopeptide ligation can be utilized for site-selective side chain labeling at an introduced Dap residue with minimal off-target labeling of Lys residues. Additionally, we generate engineered peptide topologies via intramolecular side chain-to-tail cross-links and produce direct protein–cyclic peptide fusions via efficient intermolecular ligation. We also show that OaAEP1-catalyzed isopeptide ligation extends to d-peptide acceptors containing a retro-inverso d-Leu-d-Dap motif. This capability further expands the range and complexity of isopeptide-linked products that can be accessed with OaAEP1, which we exemplify by forming a hybrid d-/l- bicyclic peptide topology where both termini are linked to internal side chains.
期刊介绍:
The flagship journal of the American Chemical Society, known as the Journal of the American Chemical Society (JACS), has been a prestigious publication since its establishment in 1879. It holds a preeminent position in the field of chemistry and related interdisciplinary sciences. JACS is committed to disseminating cutting-edge research papers, covering a wide range of topics, and encompasses approximately 19,000 pages of Articles, Communications, and Perspectives annually. With a weekly publication frequency, JACS plays a vital role in advancing the field of chemistry by providing essential research.