Characterization of an Iterative Halogenase Acting on Ribosomal Peptides Underlies the Combinatorial Biosynthesis Logic of Lasso Peptides

Halogenation is commonly utilized in medicinal chemistry
for the
improvement of drug leads. Flavin-dependent halogenases (FDHs) are
ubiquitous across all domains of life, yet iterative FDHs are rare
in the biosynthesis of ribosomally synthesized and post-translationally
modified peptides (RiPPs). Herein, we characterize a novel iterative
FDH, ChlH, which orchestrates nonsequential chlorination of two specific
Trp within the core peptide of a lasso precursor containing three
Trp. Biochemical and computational studies enable the characterization
of ChlH, which employs unique protein-peptide interactions (PPIs)
between its distinct N- and C-terminal motifs and a crucial recognition
sequence (RS-II) downstream of RS-I in the leader peptide. Previous
studies have demonstrated the indispensability of RS-I for lasso peptide
biosynthesis, while RS-II was considered to be replaceable. Furthermore,
we find that the core peptide substantially contributes to the PPI.
Bioinformatic analysis reveals the prevalence of homologous FDHs in
the biosynthetic gene clusters (BGCs) of various RiPP classes. Heterologous
expression of the chl BGC yields non-, mono-, and
dichlorinated lasso peptides, with chlorination, particularly dichlorination,
enhancing their antibacterial activity. This study expands the FDH
activity spectrum to include iterative catalysis on ribosomal peptides
and underscores the significance of RS-II in tailoring enzymes for
the combinatorial biosynthesis of lasso peptides.