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Title:Revealing the molecular details of azoline formation in ribosomal natural product biosynthesis
Author(s):Dunbar, Kyle
Director of Research:Mitchell, Douglas A.
Doctoral Committee Chair(s):Mitchell, Douglas A
Doctoral Committee Member(s):van der Donk, Wilfred A.; Nair, Satish K.; Gerlt, John A.
Department / Program:Chemistry
Discipline:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Azoline Biosynthesis
ribosomally synthesized and post-translationally modified peptide (RiPP) Biosynthesis
thiazole/oxazole-modified microcin (TOMM) Biosynthesis
Cyclodehydratase
YcaO
Abstract:The thiazole/oxazole-modified microcins (TOMMs) are a recently grouped class of ribosomally synthesized and posttranslationally modified peptides. Encoded by many bacteria and archaea, these natural products occupy a large chemical and functional space and are linked by the presence of the eponymous azole moieties. Previous work has demonstrated that an evolutionarily conserved, heterotrimeric enzyme complex (TOMM synthetase) is responsible for azole biogenesis. The TOMM synthetase transforms select serine, threonine and cysteine residues in the core peptide into azole heterocycles via a two-step cyclodehydration-dehydrogenation reaction. The installation of the azol(in)e heterocycles endows the peptide with rigidity and, in all characterized TOMM natural products, is required for biological activity. Despite being reconstituted nearly two decades ago, many questions remain regarding the role of each protein in the TOMM synthetase and the mechanism of cyclodehydration. Previous attempts to elucidate the molecular details of azole formation were impeded by the poor stability/solubility of reconstituted TOMM synthetases. In this thesis I report the characterization of the biosynthetic machinery from two novel TOMM cluster from Bacillus sp. Al Hakam that do not suffer from these issues. Using these robust synthetase complexes, I demonstrate that the cyclodehydration reaction proceeds via a phosphorylated hemi-orthoamide intermediate (chapter 2) and provide definitive roles from each of the proteins in the cyclodehydratase complex (chapter 4 and 5). My findings identify an unprecedented role for ATP and provide the first function to the previously uncharacterized YcaO superfamily of proteins. Furthermore, in order to complete my mechanistic investigations of the TOMM synthetase, I developed a novel strategy for generating peptides with site-specific carbonyl oxygen isotope labels (chapter 3). I also demonstrate that this strategy can be expanded to the site-specific installation of thioamides.
Issue Date:2015-01-21
URI:http://hdl.handle.net/2142/73109
Rights Information:Copyright 2014 Kyle L. Dunbar
Date Available in IDEALS:2015-01-21
Date Deposited:2014-12


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