School of Biological Sciences, Nanyang Technological University, Singapore, Singapore; School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing, China.
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore.
J Biol Chem. 2023 Mar;299(3):102997. doi: 10.1016/j.jbc.2023.102997. Epub 2023 Feb 9.
Plant legumains are Asn/Asp-specific endopeptidases that have diverse functions in plants. Peptide asparaginyl ligases (PALs) are a special legumain subtype that primarily catalyze peptide bond formation rather than hydrolysis. PALs are versatile protein engineering tools but are rarely found in nature. To overcome this limitation, here we describe a two-step method to design and engineer a high-yield and efficient recombinant PAL based on commonly found asparaginyl endopeptidases. We first constructed a consensus sequence derived from 1500 plant legumains to design the evolutionarily stable legumain conLEG that could be produced in E. coli with 20-fold higher yield relative to that for natural legumains. We then applied the ligase-activity determinant hypothesis to exploit conserved residues in PAL substrate-binding pockets and convert conLEG into conPAL1-3. Functional studies showed that conLEG is primarily a hydrolase, whereas conPALs are ligases. Importantly, conPAL3 is a superefficient and broadly active PAL for protein cyclization and ligation.
植物类半胱氨酸天冬氨酸蛋白酶是天冬氨酸/谷氨酸特异性内切蛋白酶，在植物中具有多种功能。肽天冬酰胺连接酶（PALs）是一种特殊的类半胱氨酸天冬氨酸蛋白酶亚型，主要催化肽键的形成而不是水解。PALs 是多功能的蛋白质工程工具，但在自然界中很少发现。为了克服这一限制，我们在这里描述了一种两步法来设计和工程基于常见的天冬酰胺内肽酶的高效、高产量的重组 PAL。我们首先构建了一个源自 1500 种植物类半胱氨酸天冬氨酸蛋白酶的共识序列，设计了进化上稳定的 conLEG，与天然类半胱氨酸天冬氨酸蛋白酶相比，其在大肠杆菌中的产量提高了 20 倍。然后，我们应用连接酶活性决定因子假说，利用 PAL 底物结合口袋中的保守残基，将 conLEG 转化为 conPAL1-3。功能研究表明，conLEG 主要是一种水解酶，而 conPALs 是连接酶。重要的是，conPAL3 是一种超高效且广泛活性的 PAL，可用于蛋白质环化和连接。
