Expanding the Chemical Space of Antimicrobial Peptides via Enzymatic Prenylation
Hikari Ozawa, Azusa Miyata, Seiichiro Hayashi, Noriyuki Miyoshi, Koichi Kato, Sohei Ito, Daisuke Fujinami
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 C δ2 and C γ 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.