Date of Award

2022

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natural Science and Mathematics, Chemistry and Biochemistry

First Advisor

John A. Latham

Second Advisor

Scott Nichols

Third Advisor

Martin Margittai

Fourth Advisor

Michelle K. Knowles

Keywords

Bioinformatics, Radical-S-adenosylmethionine enzyme, Ribosomally synthesized and posttranslationally modified peptide, Sequence similarity network, TIG biosynthesis

Abstract

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 tigABCDEF, found in the bacterium Paramaledivibacter caminithermalis. 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.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Yi Lien

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

65 pgs

Discipline

Biochemistry, Chemistry

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