Characterization of Cyclopropyl Synthases Involved in the Maturation of Ribosomally Synthesized and Posttranslationally Modified Peptides

<p>Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of natural products with significant human health implications. RiPPs are synthesized from a genetically encoded precursor peptide that undergoes significant modifications by maturing enzymes, or maturases. Recently, radical-S-adenosylmethionine (rSAM) enzymes have emerged as an important family of RiPP maturases. rSAM enzymes have been shown to install ether, thioether, and carbon-carbon bonds on the precursor peptide. These modifications usually define the backbone structure of the mature RiPP. This thesis describes the characterization of a novel RiPP modification catalyzed by the radical S-adenosylmethionine enzyme TigE. TigE belongs to the TIG biosynthetic gene cluster (BGC), which is encoded by <em>tigABCDEF</em>, found in the bacterium <em>Paramaledivibacter caminithermalis</em>. The TIG precursor peptide, TigB, is comprised of a repeating TIGSVSG motif. Using a variety of chromatography, mass spectrometry, isotopic labeling, and NMR spectroscopy techniques, we show that TigE catalyzes the formation of C-C bond between the γ-carbons on TigB isoleucine residues (Ile), forming a methyl-cyclopropyl glycine (mCPG). Using crystallography and site-directed mutagenesis, we also revealed that the TigE residue Tyr339 is critical for both the coordination of iron-sulfur clusters and chemistry and could be used as a biomarker for the discovery of other cyclopropyl synthases. This novel RiPP modification provided a reaction scope of radical S-adenosylmethionine enzymes.</p>

Name thesis:degree_name

M.S.

Level thesis:degree_level

Masters Thesis

Year dc:date.available

2022

Author dc:creator

• Lien, Yi

Contributors dc:contributor

• John A. Latham
• Scott Nichols
• Martin Margittai
• Michelle K. Knowles

• Bioinformatics
• Radical-S-adenosylmethionine enzyme
• Ribosomally synthesized and posttranslationally modified peptide
• Sequence similarity network
• TIG biosynthesis
• Biochemistry
• Biochemistry, Biophysics, and Structural Biology
• Chemistry
• Other Chemistry

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

en

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2129

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3127