On the biosynthesis and discovery of ribosomally synthesized and post-translationally modified peptides

Thiazole/oxazole-modified microcins (TOMMs) comprise a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) that all contain thiazole and oxazole heterocycles derived from cysteine, serine, and threonine residues, respectively. The thiazole/oxazole heterocycle is installed over two distinct steps. First, the cyclodehydratase cyclizes an unmodified cysteine, serine, or threonine residue to a thiazoline or oxazoline heterocycle, which can be oxidized by a FMN dependent dehydrogenase to afford a thiazole or oxazole heterocycle. The biosynthesis of a number of TOMMs has been investigated (Chapter 1), yet a number of fundamental questions regarding substrate processing remained. Genome mining efforts revealed an uncharacterized TOMM gene cluster in Bacillus sp. Al Hakam (Balh) that contained substrates and enzymes that ultimately overcame previous limitations. Initial reconstitution of the enzymes revealed cyclization of half of the heterocyclizable residues. This observation inspired a series of experiments to explain the observed selectivity. Using a combination of high-resolution mass spectrometry, site-directed mutagenesis, and kinetics, I found that the location of a heterocyclizable residue and the presence of a preceding glycine largely dictate heterocycle formation, which uniquely proceeds in an overall C- to N-terminal fashion (Chapter 2). While characterizing the cyclodehydratase, it became evident that omission of the dehydrogenase had no effect on cyclodehydratase activity. This provided the opportunity to separate the function of the two enzymes and investigate how the dehydrogenase interacts with the cyclodehydratase and processes substrate (Chapter 3). Many of the tools I developed to study the Balh TOMM synthetase proved useful for structural and biosynthetic studies of other RiPPs (Appendix A)