Examining the Leader Peptide Recognition Domain in the Lasso Peptide Biosynthetic Pathway

Abstract

Biopolymers, such as proteins, are one of the many possible scaffolds leading to promising natural products. One class of natural products, ribosomally synthesized and posttranslationally modified peptides (RiPPs), has various subclasses that share similarities in their enzymatic machinery; specifically, the precursor peptide recognition element (RRE) is a homologous domain found within enzymes of several RiPP subclasses. Lasso peptides, one subclass of RiPPs, are unique in their lariat structure, including the threading of the tail through a ring. Lasso peptide biosynthetic clusters either have the RRE as a stand-alone protein known as B1, or have the RRE as a domain, called “B1” here, of a full-length B enzyme. The full-length B enzyme’s second domain, called “B2” here, is a serine protease, which exists as a stand-alone protein B2 when the RRE is a stand-alone protein. With the RRE within lasso peptides as a launching point for this study, this work examined the possible transposable functions of the two domains of the B enzyme within the astexin-1 and astexin-3 systems. The replacement of the cognate “B1” domain and the leader peptide of the astexin-3 system with their corresponding noncognates from the astexin-1 system resulted in low levels of production of cognate lasso peptide. Additionally, the replacement of the cognate “B1” domain and the entire precursor of the astexin-3 system with their corresponding noncognates from the astexin-1 system resulted in the production of truncated linear core peptide. Thus, this study supported that the functions of the two domains are indeed transposable; the results represent a step towards gaining control of the biosynthetic machinery of lasso peptides. This work also included phylogenetic and sequence similarity-based analysis of the “B1” domain within lasso peptides and compared this to the 16s rRNA-derived phylogeny, thereby confirming previous classifications of lasso peptides and also serving as an inquiry into the connections amongst various RiPPs subclasses.