Arginine-<i>N,N</i>′-bisprenyltransferases: Switchable Catalysis in Consecutive Guanidine-<i>N</i>-prenylation

Lipidation is a promising strategy to enhance the membrane affinity and the serum stability of peptide drugs. Cyanobactin prenyltransferases catalyze the prenylation of peptides with diverse chemoselectivity. Among these enzymes, AutF and AgcF catalyze the mono- and bisprenylations of Arg-<i>N</i>^ω, respectively, although the structural basis for these distinct prenylation modes remained unknown. Through genome mining, we herein identified a new Arg-<i>N</i>^ω-bisprenyltransferase, DciF, from <i>Dolichospermum circinale</i> AWQC310F. Crystallographic analysis and subsequent mutagenesis studies identified key active site residues that play pivotal roles in Arg-<i>N</i>^ω-bisprenylation. Manipulations of the active site pocket successfully converted an Arg-<i>N</i>^ω-bisprenyltransferase into an Arg-<i>N</i>^ω-monoprenyltransferase, and <i>vice versa</i>, elucidating its role as a determinant factor for prenylation rounds. DciF efficiently catalyzed Arg-<i>N</i>^ω-bisprenylation on various cyclic and linear peptides, demonstrating its remarkable potential as a biocatalytic tool for site-selective peptide modification. This study broadens the scope of biocatalytic peptide modifications and establishes a new framework for engineering cyanobactin prenyltransferases to control their prenylation rounds.