Enzymatic Reconstitution and Biosynthetic Investigation of the Lasso Peptide Fusilassin

Abstract

Lasso peptides are a class of ribosomally synthesized 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 remain poorly characterized due to 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 branchedcyclic 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 an important protein-protein interaction site, illustrating how genomics 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.

Factors Governing the Thermal Stability of Lasso Peptides

“…Since the study of the heat sensitivity of caulosegnins I and III, many newly isolated lasso peptides have been tested for thermal stability; this led to the discovery of a significant number of class II lasso peptides that can unthread at elevated temperatures –. The occurrence of heat‐sensitive lasso peptides is probably not linked to a biological function, as the studied lasso peptides were usually isolated from strains living at temperature ranges at which thermally induced unthreading would either not occur or occur only very slowly.…”

“…The occurrence of heat‐sensitive lasso peptides is probably not linked to a biological function, as the studied lasso peptides were usually isolated from strains living at temperature ranges at which thermally induced unthreading would either not occur or occur only very slowly. Nonetheless, the thermal stability of these compounds is an interesting feature to study and can be used to prove the lasso topology of newly isolated members of this natural product family . In addition, it is necessary to know the stability of a lasso peptide at high temperatures when planning to use it for certain applications, employ it for certain chemical transformations, or establish isolation protocols (e.g., it is not advisable to use thermal denaturation to precipitate proteins during the work‐up of a heat‐sensitive lasso peptide).…”

“…The most straightforward approach was described above for the discovery of this behavior for caulosegnins I and III. Concretely, the lasso peptide in question is incubated for different durations at different temperatures and then analyzed by means of liquid chromatography–mass spectrometry (LC‐MS) …”

“…In contrast, the lower plug substitution F17W in caulonodin IV (Ser1–Glu9 ring, 29 atoms) confers thermal stability . Considering these observations in the light of other thermally stable lasso peptides with Xxx1–Glu9 rings (Table ), it becomes apparent that ring size alone is not enough to predict the heat stability of a lasso peptide. However, a general trend is that the larger the macrolactam ring, the harder it is to maintain the lasso fold, especially at elevated temperatures.…”

“…The rigid lasso fold can confer high stability against proteolytic degradation and many lasso peptides are known to withstand prolonged incubation at elevated temperatures . For class II lasso peptides, heat stability is a remarkable feat, as their topology is only maintained by steric means.…”