Functional and mechanistic elucidation of peptide backbone thioamidation

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2021

Author dc:creator

• Liu, Andi

Contributors dc:contributor

• Mitchell, Douglas A
• Imlay, James A
• Cronan, John E
• van der Donk, Wilfred A

• thioamide
• RiPPs
• YcaO
• posttranslational modification

Statement dc:rights

• Copyright 2021 Andi Liu

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/110774

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/110774