INVESTIGATION OF THE MATURATION AND DEGRADATION OF LASSO PEPTIDES THROUGH IN VITRO STUDIES

Abstract

Lasso peptides are a class of small molecules characterized by a unique threaded structure. In addition to this shape, lasso peptides are also of interest due to their range of properties, ranging from narrow spectrum anti-microbial to inhibiting viral envelope fusion. In recent years, with the increase in genomes available, it has become apparent that lasso peptides are present in a wider range of bacteria than had been identified through traditional natural products screening. In Chapters 2, a gene cluster containing two lasso peptides from the freshwater bacterium Asticcacaulis excentricus was examined to learn more about the promiscuity of lasso peptide related enzymes. The gene cluster contained a novel enzyme, termed a lasso isopeptidase, which can turn lasso peptides into a linear form. Using alanine scanning of the precursor peptides, it was determined that both the maturation enzymes as well as the isopeptidase were able to process a range of substrates. In Chapter 3, the structure of the isopeptidase enzyme was solved with and without precursor to learn more about how it recognized its substrates. In addition, the isopeptidase sequence was used to identify novel lasso peptide gene clusters, further expanding the known examples for this class. In Chapter 4, a novel lasso peptide gene cluster from Thermobifida fusca, a thermophilic bacterium, was identified as a source of soluble maturation enzymes for lasso peptide biosynthesis. The product was produced both through heterologous expression in E. coli, as well as by incubation of the precursor peptide with the maturation enzymes in vitro. This system was used to determine for the first time information about lasso peptide biosynthetic rates. In Chapter 5, a lasso peptide gene cluster was identified from Thermobifida cellulosilytica with a previously unidentified O-methyltransferase within the cluster. In addition, a similar enzyme was also identified within an unexplored class of products called actinopeptides. Each of these systems was explored through a combination of heterologous expression and in vitro experiments. Through the course of these studies, lasso peptide biosynthesis and degradation are on a more solid foundation for future examination.