Screening and Engineering of Ancestral Sortase Es That Enable High-Fidelity Enzyme Design with Activity Trade-Off

Protein grafting techniques that enable conjugation of diverse compounds to target proteins endow them with new functions. Sortase-mediated ligation (SML) is one such method in which sortases catalyze transpeptidation between a substrate protein or peptide bearing a cell wall sorting signal (CWSS) and a nucleophile containing an N-terminal triglycine motif. In this study, we identified novel sortase Es suitable for SML by combining in silico enzyme screening with ancestral sequence reconstruction (ASR). Eight ancestral sortases Es (1AcSE-8AcSE) were designed, of which four (1AcSE, 2AcSE, 3AcSE, and 8AcSE) were expressed in soluble form and exhibited moderate thermal stability. HPLC analysis using Ac-YNL(A/P)ETGA and GGGKY peptides revealed that 2AcSE displayed no activity toward Ac-YNLPETGA, suggesting its unique substrate specificity. Structural analysis of 2AcSE indicated that differences in Loop A, which corresponds with the β3-β4 loop, may contribute to its specificity. We designed ΔAcSE5, a variant of a previously characterized high-activity AcSE5, by replacing its Loop A (residues 56'-61', GEAPLK) with the shorter TG motif from 2AcSE. ΔAcSE5 exhibited improved specificity for Ac-YNLAETGA over Ac-YNLPETGA, with a trade-off in activity and reduced byproduct formation during conjugation of a shark antibody to GGG-Venus. These findings demonstrate that database-driven screening and structure-function analysis of AcSEs can guide the design of novel sortase E variants optimized for SML applications.