School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.
Chem Soc Rev. 2022 May 23;51(10):4121-4145. doi: 10.1039/d0cs01148g.
Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.
定点蛋白质修饰是一种广泛应用的生化工具。然而，蛋白质修饰技术的发展存在许多挑战，特别是要实现定点性、反应效率和多功能性。肽连接酶及其底物的工程化已被用于解决这些挑战。本综述将重点介绍天冬酰胺酰基肽连接酶（sortase）、肽基天冬氨酸连接酶（PALs）和枯草杆菌蛋白酶的变体；详细说明它们固有的特异性如何用于定点蛋白质修饰。本综述将探讨这些酶和底物的工程化如何导致反应效率的提高，主要是由于催化活性的增强和可逆性的降低。它还将描述如何通过工程化肽连接酶来拓宽其底物范围，为扩展生化工具包开辟新的机会，特别是通过开发使用正交肽连接酶将多个底物定点连接到蛋白质上的技术。
