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Title:Functional and mechanistic elucidation of peptide backbone thioamidation
Author(s):Liu, Andi
Director of Research:Mitchell, Douglas A
Doctoral Committee Chair(s):Mitchell, Douglas A
Doctoral Committee Member(s):Imlay, James A; Cronan, John E; van der Donk, Wilfred A
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
posttranslational modification
Abstract:YcaO enzymes are best known to catalyze the ATP-dependent peptide backbone activation and subsequent formation of azoline heterocycles. It has been proposed that YcaO enzymes can also install lactamidines and thioamides onto peptides through similar mechanisms. These modifications are found in a class of natural products named ribosomally synthesized and post-translationally modified peptides (RiPPs) and are critical for their biological functions. In addition to RiPPs, thioamide has also been observed as a post-translational modification in two essential protein complexes: methyl-coenzyme M reductase (MCR) and the ribosome. Genetic studies in the methanogen Methanosarcina acetivorans have linked the thioamide in MCR with ycaO and its neighboring gene tfuA, which has no known function. In this thesis, I report the in vitro thioamidation of an MCR-derived peptide using methanogenic YcaOs and inorganic sulfide (chapter II). The substrate specificity of the YcaO was investigated and the interaction with the substrate peptide was established. Leveraging the crystal structure of a methanogenic YcaO bound to its MCR peptide, I identified and tested the residues involved in substrate binding and catalysis through site-directed mutagenesis (chapter III). Further, the function of the TfuA protein was identified: TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO towards the thioamidation substrate (chapter IV). My biophysical and mutational analyses of TfuA also uncovered its binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser/Lys catalytic pair.
Issue Date:2021-03-02
Rights Information:Copyright 2021 Andi Liu
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05

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