Modulating the Acceptor Preference of His-C-Geranyltransferase LimF

Lipidation stands as a pivotal strategy for enhancing the metabolic stability of target peptides. Prenyltransferases in cyanobactin biosynthesis have garnered significant attention as potential peptide lipidation biocatalysts because of their exceptional regio- and chemoselectivity. However, these enzymes often exhibit a biased preference for certain acceptor substrates, requiring specific amino acids adjacent to the modifying residue. In this study, we demonstrate the structure-guided engineering of LimF, a His-C-geranyltransferase, to broaden its peptide substrate tolerance. By altering key residues in the peptide-binding pocket, we created a LimF variant capable of modifying sequence motifs previously inaccessible to the wildtype enzyme. The variant successfully modified some previously unfavored sequence motifs in artificial peptide substrates and bioactive peptide agents, validating the engineered substrate scope. With the discovery of novel peptide prenyltransferases, this approach would lead to a more comprehensive toolbox of peptide prenylation biocatalysts.