Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester, UK.
Faraday Discuss. 2024 Sep 11;252(0):295-305. doi: 10.1039/d4fd00019f.
Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue -methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRS) for encoding MeHis into proteins, by transplanting five known active site mutations from (PylRS) into the single domain PylRS from ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the system, G1PylRS can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRS is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.
遗传密码扩展已成为酶设计和工程的强大工具，为复杂的催化机制提供了新的见解，并能够开发具有新催化功能的酶。在这方面，非典型组氨酸类似物 -甲基组氨酸（MeHis）因其能够作为金属配位配体或催化亲核试剂发挥作用而具有多功能性，其反应模式类似于小分子催化剂，如 4-二甲氨基吡啶（DMAP）。在这里，我们通过将五个已知的活性位点突变从 （PylRS）移植到来自 ISO4-G1 的单结构域 PylRS 中，开发了一种高效的用于将 MeHis 编码到蛋白质中的氨酰 tRNA 合成酶（G1PylRS）。与 系统中需要高浓度 MeHis（5-10 mM）相比，G1PylRS 可以在约 0.1 mM 的 MeHis 浓度下有效运行，从而可以更经济地生产一系列高浓度的含有 MeHis 的酶。有趣的是，G1PylRS 也是一种“多特异性”氨酰 tRNA 合成酶（aaRS），能够掺入包括 3-吡啶基丙氨酸和 2-氟苯丙氨酸在内的五种不同的非典型氨基酸（ncAAs）。这项研究为可扩展生产含有非典型氨基酸（如 MeHis）作为关键催化元件的工程酶提供了重要步骤。
