澳大利亚昆士兰大学David Craik研究小组重新利用植物肽环化酶实现了靶向赖氨酸酰化。相关论文于2024年5月24日发表在《自然—化学》杂志上。

研究人员表示，转肽酶是蛋白质工程的有力工具，但在很大程度上仅限于作用于蛋白质的骨干末端。用于内部蛋白质标记的替代酶方法需要大量的识别基序或非蛋白质原性反应伙伴，这可能限制了可以修饰的蛋白质或可以安装的修饰类型。

在该研究中，研究小组通过重新利用一种工程天冬酰胺连接酶，开发了一种标记赖氨酸侧链ε-胺的策略，该酶天然催化肽头到尾环化，用于与肽底物兼容的多功能异肽连接。该研究团队发现与亮氨酸残基相邻的内部赖氨酸类似于传统的N端甘氨酸亮氨酸底物。

这种二肽基序通过内部赖氨酸侧链实现有效的分子内或分子间连接，最小化地余下通过异肽键连接到赖氨酸侧链的天冬酰胺C端。这种方法的多功能性通过化学酶合成具有非天然C-端到侧链拓扑结构的肽，以及将化学修饰的肽偶联到重组蛋白上得到了证明。

附：英文原文

Title: Repurposing a plant peptide cyclase for targeted lysine acylation

Author: Rehm, Fabian B. H., Tyler, Tristan J., Zhou, Yan, Huang, Yen-Hua, Wang, Conan K., Lawrence, Nicole, Craik, David J., Durek, Thomas

Issue&Volume: 2024-05-24

Abstract: 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.

DOI: 10.1038/s41557-024-01520-1

Source: https://www.nature.com/articles/s41557-024-01520-1