Abstract：
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.
摘要：
植物豆科是Asn/Asp特异性内肽酶（AEP），在植物中具有多种功能。肽天冬酰胺基连接酶（PAL）是一种特殊的豆科蛋白酶亚型，主要催化肽键形成而不是水解。PAL是通用的蛋白质工程工具，但在自然界中很少发现。为了克服这个限制,在这里，我们描述了一种两步方法来设计和设计一种基于常见AEP的高产高效重组PAL。我们首先构建了源自1,500个植物豆科的共有序列，以设计进化上稳定的豆科蛋白conLEG，该序列可以在大肠杆菌中产生，相对于天然豆科蛋白，产量高20倍。然后，我们应用LAD（连接酶活性决定子）假设来利用PAL底物结合袋中的保守残基，并将conLEG转换为conPAL1-3。功能研究表明，conLEG主要是一种水解酶，而conPAL是连接酶。重要的是,conPAL3是用于蛋白质环化和连接的超高效和广泛活性的PAL。
