Enzymatic Prenylation of Proteins and Peptides: From Cysteine S ‐Prenylation to Tryptophan‐Selective Biocatalysis

Posttranslational prenylation endows polypeptides with lipophilic properties, facilitating essential biological functions such as membrane targeting and signaling. Unlike linear fatty acyl groups, the branched and unsaturated structure of isoprenoids introduces complex sterics and restricted conformational degrees of freedom. These unique features enable the fine-tuning of membrane behaviors, including lateral partitioning, packing defects, and hydrophobic mismatches, which can dictate biological outcomes. Focusing on biocatalytic prenylation, this review summarizes eukaryotic, cyanobactin, and trans-isoprenyl diphosphate synthase (trans-IDS)-like prenyltransferases. We examine how active-site motifs govern specificity for both the amino acid acceptor and the donor isoprenoid, thereby enabling the use of longer-chain variants and geometric isomers, as well as regioselective modification of tryptophan. Furthermore, we compare the biophysical properties of emerging tryptophan C-prenylation with those of conventional cysteine S-prenylation and linear fatty acylation, highlighting their distinct roles in modulating polypeptide function and behavior. Finally, we focus on applications of enzymatic prenylation in antimicrobial development and protein engineering, outlining future opportunities for AI-guided design of prenylated peptides and proteins.