TY - JOUR
T1 - Enzymatic Reconstitution and Biosynthetic Investigation of the Lasso Peptide Fusilassin
AU - Dicaprio, Adam J.
AU - Firouzbakht, Arash
AU - Hudson, Graham A.
AU - Mitchell, Douglas A.
N1 - Publisher Copyright:
© 2018 American Chemical Society.
PY - 2019/1/9
Y1 - 2019/1/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85059778124&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85059778124&partnerID=8YFLogxK
U2 - 10.1021/jacs.8b09928
DO - 10.1021/jacs.8b09928
M3 - Article
C2 - 30589265
AN - SCOPUS:85059778124
SN - 0002-7863
VL - 141
SP - 290
EP - 297
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 1
ER -