Enzymatic Reconstitution and Biosynthetic Investigation of the Lasso Peptide Fusilassin

Lasso
peptides are a class of ribosomally synthesized and post-translationally
modified natural product which possess a unique lariat knot conformation.
The low entropy “threaded” conformation endows lasso
peptides with considerable resistance to heat and proteolytic degradation,
which are attractive properties for the development of peptide-based
therapeutics. Despite their discovery nearly 30 years ago, the molecular
mechanism underlying lasso peptide biosynthesis remains poorly characterized
due to the low stability of the purified biosynthetic enzymes. Here,
we report the biosynthetic reconstitution of a lasso peptide derived
from Thermobifida fusca, termed fusilassin. Beyond
robust catalytic activity, the fusilassin enzymes demonstrate extraordinary
substrate tolerance during heterologous expression in E. coli and upon purification in cell-free biosynthetic reconstitution reactions.
We provide evidence that the fusilassin biosynthetic enzymes are not
capable of forming branched-cyclic products but can produce entirely
unrelated lasso peptides. Finally, we leveraged our bioinformatic
survey of all lasso peptides identified in GenBank to perform coevolutionary
analysis of two requisite biosynthetic proteins. This effort correctly
identified residues governing an important protein−protein
interaction, illustrating how genomic insight can accelerate the characterization
of natural product biosynthetic pathways. The fusilassin enzymes described
within represent a model system for both designing future lasso peptides
of biomedical importance and also for elucidating the molecular mechanisms
that govern lasso peptide biosynthesis.