Structural investigation of lanthipeptides involved in substrate controlled pathways and methods for leader peptide removal

Ribosomally synthesized and post-translationally modified peptides are a family of natural products with diverse activities, with lanthipeptides as one of the most prominent classes. The diverse ring topology of lanthipeptides is formed by four classes of enzymes with substrate tolerance that holds potential for development of biotechnological techniques for macrocyclizations that may increase the utility of peptide therapeutics. The general biosynthetic scheme of a leader peptide that plays a role in the processing of the core peptide by biosynthetic enzymes is powerful as such pathways could potentially be used to make products that are very different from the natural product. This strategy would be most successful when the structural characterization and mechanism of lanthionine synthetases is fully understood. However, a difficult obstacle for high throughput studies of these natural products is removal of the leader peptide. Chapter 2 discusses the development of a general method using unnatural amino acid incorporation to replace the peptide bond between the leader peptide and the core peptide with an ester bond, which can be easily hydrolyzed in basic environments. This proof-of-concept method was site selective for the tested substrates setting the stage for further development and optimization for increasing the yields of peptides produced by this route. Chapter 3 extends this theme through the identification of a general protease for the removal of double glycine motif-leader peptides, used in a subset of RiPPs which had escaped the development of a general method for leader peptide removal. The cognate proteases in lanthipeptide biosynthetic machineries are often membrane associated proteins that suffer from a decrease in catalytic activity when taken out of their cellular environment and/or that are not active at if segregated from the membrane-bound domains. The structural characterization of an isolated protease domain of a LanT-type transporter from Lachnospiraceae C6A11 with a substrate mimic offers molecular understanding of the recognition by this type of proteases. In addition, the chapter explores the substrate scope of this protease. The range of substrates tested constitutes only a sliver of the approximately 1500 peptides that contain a cleavage motif that may be successfully processed by this protease as suggested by the mutagenesis and proteolytic studies covered here. Chapter 4 presents the three dimensional structural elucidation by NMR spectroscopy of seven lanthipeptides found to be involved in LanM-catalyzed modifications where substrate control appears to play an important role in the outcome of enzyme catalysis. This study characterizes for the first time five prochlorosins, which adopt different globular structures, as well as the two component cytolysin peptides, which adopt helical conformations consistent with traits previously shown to be important in membrane permeabilization by other helical peptides. These peptides were chosen because previous studies showed that the CylM-catalyzed formation of the cytolysins is dictated by the amino acid sequence of the substrate and biased toward the formation of rare LL-lanthionine stereochemistry. The prochlorosin peptides were chosen because they are all modified by one lanthionine synthetase yet they have very different amino acid sequences, suggesting that there may be elements of substrate control in the biosynthesis of these marine lanthipeptides. Their three-dimensional structures had not yet been elucidated. Furthermore, this class of peptides seems to have widespread presence in marine cyanobacteria with their lanthionine synthetases under strong selection pressure to accept a wide array of precursor peptide sequences, suggesting that their structural diversity may be important in an ecological context. This hypothesis is yet to be directly tested