Consensus design and engineering of an efficient and high-yield peptide asparaginyl ligase for protein cyclization...

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.