| Summary: | Plant legumains are Asn/Asp-specific endopeptidases (AEPs) 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 AEPs. We first constructed a
consensus sequence derived from 1,500 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 LAD (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
super-efficient and broadly active PAL for protein cyclization and ligation.
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