Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing to a locked three-dimensional structure, lasso peptides can be unusually stable toward heat and proteolytic degradation. Some lasso peptides have been shown to bind human cellsurface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.
Benzoxaboroles are a class of boron-containing compounds with a broad range of biological activities. A subset of benzoxaboroles have antimicrobial activity due primarily to their ability to inhibit leucyl-tRNA synthetase (LeuRS) via the oxaborole tRNA trapping mechanism, which involves formation of a stable tRNA Leu-benzoxaborole adduct in which the boron atom interacts with the 2′-and 3′-oxygen atoms of the 3′-terminal tRNA adenosine. We sought to identify other antibacterial targets for this promising class of compounds by means of mode of action studies, and we selected a nitrophenyl sulfonamide-based oxaborole (PT638) as a probe molecule because it had potent antibacterial activity (MIC of 0.4 μg/mL against methicillin-resistant Staphylococcus aureus) but did not inhibit LeuRS (IC 50 > 100 μM). Analogues of PT638 were synthesized to explore the importance of the sulfonamide linker and the impact of altering the functionalization of the phenyl ring. These structure-activity relationship studies revealed that the nitro substituent was essential for activity. To identify the target for PT638, we raised resistant strains of S. aureus and whole genome sequencing revealed mutations in leuRS, suggesting that the target for this compound was indeed LeuRS, despite the lack of enzyme inhibition. Subsequent analysis of PT638 metabolism demonstrated that bacterial nitroreductases readily converted this compound into the amino analogue, which inhibited LeuRS with an IC 50 of 3.0 ± 1.2 μM demonstrating that PT638 is thus a prodrug.
The RiPP precursor recognition element (RRE) is a conserved domain found in many prokaryotic ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthetic gene clusters (BGCs). RREs bind with high specificity and affinity to a recognition sequence within the N-terminal leader region of RiPP precursor peptides. Lasso peptide biosynthesis involves an RRE-dependent leader peptidase, which is discretely encoded or fused to the RRE as a di-domain protein. Here we leveraged thousands of predicted BGCs to define the RRE:leader peptidase interaction through evolutionary covariance analysis. Each interacting domain contributes a three-stranded β-sheet to form a hydrophobic β-sandwich-like interface. The bioinformaticsguided predictions were experimentally confirmed using proteins from discrete and fused lasso peptide BGC architectures. Support for the domain−domain interface derived from chemical shift perturbation, paramagnetic relaxation enhancement experiments, and rapid variant activity screening using cell-free biosynthesis. Further validation of selected variants was performed with purified proteins. We developed a p-nitroanilide-based leader peptidase assay to illuminate the role of RRE domains. Our data show that RRE domains play a dual function. RRE domains deliver the precursor peptide to the leader peptidase, and the rate is saturable as expected for a substrate. RRE domains also partially compose the elusive S2 proteolytic pocket that binds the penultimate threonine of lasso leader peptides. Because the RRE domain is required to form the active site, leader peptidase activity is greatly diminished when the RRE domain is supplied at substoichiometric levels. Full proteolytic activation requires RRE engagement with the recognition sequence-containing portion of the leader peptide. Together, our observations define a new mechanism for protease activity regulation.
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