Expanding the Chemical Space of Antimicrobial Peptides via Enzymatic Prenylation.

Abstract

Antimicrobial peptides act primarily at the bacterial membrane interface. We report a biocatalytic strategy that enhances their potency by up to 18-fold. The improvement results from the enzymatic installation of bulky isoprenoid chains, which strengthens peptide-membrane interactions and promotes membrane destabilization. We characterize PalQ, an isoprenoid synthase-related prenyltransferase that is uniquely amenable to enzyme engineering. PalQ catalyzes prenylation at both N- and C-terminal tryptophan residues via positionally distinct Cδ2 and Cγ alkylation, respectively. Structure-guided mutagenesis of the prenyl donor pocket, combined with glycine substitutions near the acceptor tryptophan, expanded PalQ's substrate scope to include diverse antimicrobial peptides and long-chain donors such as geranylgeranyl diphosphate. A computationally optimized PalQ variant further improved performance under high-salt and organic solvent conditions, enabling late-stage modification of poorly soluble peptides. These results establish PalQ as a versatile platform for site-selective lipidation and expand the accessible chemical space for peptide and protein engineering.