Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs)

Abstract

Asparaginyl endopeptidases (AEPs) are thiol proteases that cleave peptide bonds after Asn/Asp(Asx) residues. However, certain AEPs also make Asx bonds and which we named peptide asparaginyl ligases (PALs). PALs, ATP-independent and stand-alone, are ideal for site-specific modifications of proteins, live cells, and biopolymers. Expanding the availability and diversity of PALs will expand tools for biotechnological and biochemical applications. In my thesis, I studied the improved production of the recombinant butelase-1, a prototype PAL, and engineering of PAL from an AEP, PeAEP. Butelase-1 was initially reported for its poor recombinant expression, and I used consensus-based engineering approach successfully to increase its recombinant expression three-fold. The second part of my thesis focuses on engineering of PeAEP into a PAL based on a hypothesis of ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. I used data mining to identify amino acids important for LADs which are located at the substrate binding pockets. They were then mutated in PeAEP to convert it successfully into a PAL. Taken together, PALs can be expanded through consensus-based engineering approach or exploiting our understanding of molecular ligase determinants of PALs for expanding the repertoire of Asx-specific ligases.