Nature Chemistry ( IF 19.2 ) Pub Date : 2024-05-24 , DOI: 10.1038/s41557-024-01520-1 Fabian B. H. Rehm , Tristan J. Tyler , Yan Zhou , Yen-Hua Huang , Conan K. Wang , Nicole Lawrence , David J. Craik , Thomas Durek
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Transpeptidases are powerful tools for protein engineering but are largely restricted to acting at protein backbone termini. Alternative enzymatic approaches for internal protein labelling require bulky recognition motifs or non-proteinogenic reaction partners, potentially restricting which proteins can be modified or the types of modification that can be installed. Here we report a strategy for labelling lysine side chain ε-amines by repurposing an engineered asparaginyl ligase, which naturally catalyses peptide head-to-tail cyclization, for versatile isopeptide ligations that are compatible with peptidic substrates. We find that internal lysines with an adjacent leucine residue mimic the conventional N-terminal glycine–leucine substrate. This dipeptide motif enables efficient intra- or intermolecular ligation through internal lysine side chains, minimally leaving an asparagine C-terminally linked to the lysine side chain via an isopeptide bond. The versatility of this approach is demonstrated by the chemoenzymatic synthesis of peptides with non-native C terminus-to-side chain topology and the conjugation of chemically modified peptides to recombinant proteins.
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重新利用植物肽环化酶进行靶向赖氨酸酰化
转肽酶是蛋白质工程的强大工具,但很大程度上仅限于作用于蛋白质主链末端。用于内部蛋白质标记的替代酶方法需要庞大的识别基序或非蛋白反应伙伴,这可能限制可以修饰的蛋白质或可以安装的修饰类型。在这里,我们报告了一种通过重新利用工程天冬酰胺酰连接酶来标记赖氨酸侧链ε-胺的策略,该酶自然催化肽头尾环化,以实现与肽底物兼容的多功能异肽连接。我们发现具有相邻亮氨酸残基的内部赖氨酸模拟了传统的 N 末端甘氨酸-亮氨酸底物。这种二肽基序能够通过内部赖氨酸侧链进行有效的分子内或分子间连接,最小限度地留下天冬酰胺 C 端通过异肽键连接到赖氨酸侧链。该方法的多功能性通过具有非天然 C 末端到侧链拓扑的肽的化学酶合成以及化学修饰的肽与重组蛋白的缀合来证明。