Asparaginyl Ligases with Engineered Substrate Specificity for Controlled, Sequential Transpeptidation Reactions.

ABSTRACT: Transpeptidases are valuable enzymes for peptide and protein engineering due to their ease of use and highly defined substrate specificities. Asparaginyl ligases are a class of highly efficient transpeptidases. Engineered asparaginyl ligases with orthogonal substrate specificities would provide access to new types of sequential transpeptidation regimes, but no such engineered variants have been reported thus far. Here, we engineer the widely used asparaginyl ligase OaAEP1 for altered substrate specificity. We find that a single amino acid substitution, Y188A, facilitates the recognition of a substrate sequence that is essentially unmodified by the parent enzyme or an alternative Y188W mutant. This orthogonality enables controlled sequential reactions for the generation of a dual N-

SUBMITTER: Zhou Y 

PROVIDER: S-EPMC12203572 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature