Tryptophan-Centric Bioinformatics Identifies New Lasso Peptide Modifications

Lasso peptides are a class of ribosomally synthesized
and post-translationally
modified peptides (RiPPs) defined by a macrolactam linkage between
the N-terminus and the side chain of an internal aspartic acid or
glutamic acid residue. Instead of adopting a branched-cyclic conformation,
lasso peptides are “threaded”, with the C-terminal tail
passing through the macrocycle to present a kinetically trapped rotaxane
conformation. The availability of enhanced bioinformatics methods
has led to a significant increase in the number of secondary modifications
found on lasso peptides. To uncover new ancillary modifications in
a targeted manner, a bioinformatic strategy was developed to discover
lasso
peptides with modifications to tryptophan. This effort identified
numerous putative lasso peptide biosynthetic gene clusters with core
regions of the precursor peptides enriched in tryptophan. Parsing
of these tryptophan (Trp)-rich biosynthetic gene clusters uncovered
several putative ancillary modifying enzymes, including halogenases
and dimethylallyltransferases expected to act upon Trp. Characterization
of two gene products yielded a lasso peptide with two 5-Cl-Trp modifications
(chlorolassin) and another bearing 5-dimethylallyl-Trp and 2,3-didehydro-Tyr
modifications (wygwalassin). Bioinformatic analysis of the requisite
halogenase and dimethylallyltransferase revealed numerous other putative
Trp-modified lasso peptides that remain uncharacterized. We anticipate
that the Trp-centric strategy reported herein may be useful in discovering
ancillary modifications for other RiPP classes and, more generally,
guide the functional prediction of enzymes that act on specific amino
acids.