Post-translational modifications catalyzed by RiPP and RiPP-adjacent enzymes

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are, as their name suggests, constructed from ribosomal peptides. The scope of biosynthetic reactions utilized by Nature to transform these peptides into structurally fascinating natural products is astounding. Leveraging these enzymes for novel uses has and will continue to provide value. However, before they can be engineered, we much first understand how these enzymes function, what their limits might be, and their natural functions. Much of the work comprising this document achieves this at various levels. Chapter 1 focuses on a class of enzymes unique to thiopeptide biosynthesis, the pyridine synthases. These multifunctional enzymes transform a heavily modified peptide into a macrocycle through generation of a pyridine ring, through a formal [4+2]-cycloaddition, concomitant with heterocycle dehydration and subsequent pyridine aromatization. Despite in vitro reconstitution of an early example of this class (TbtD), mutagenic scanning, binding data, X-ray crystallographic data, and more, it remained unclear how exactly this enzyme performs its role in thiopeptide biosynthesis. Through several routes of inquiry, including chemical cross-linking and chemical rescue, we determined that pyridine synthases use an active site Tyr residue to perform aromatization on peptide scaffolds, separating pyridine-based thiopeptides from (dehydro)piperidine-based thiopeptides. This work will inform the future use of this enzyme class in engineering pyridine-based macrocycles for biotechnological and medical applications, as well as in the structural prediction of novel thiopeptides from their biosynthetic gene clusters. Chapter 2 covers the in vitro reconstitution of a remarkably effective flavin-dependent Trp halogenase, ChlH. ChlH normally functions in the biosynthesis of the lasso peptide chlorolassin, in which two of three Trp residues in the final product are modified. Much to our surprise, ChlH modified the linear precursor peptide rather than the mature lasso scaffold. We further endeavored to push this enzyme to its limits, modifying several other peptides, RiPP natural products, and proteins. Extensive in vitro mutagenesis was performed to provide additional insight into the scope of this enzyme. ChlH holds strong potential for use in biocatalysis, among other applications where selective Trp chlorination and/or bromination may be required. Chapter 3 explores a relatively unexpected post-translational modification in biology: backbone thioamidation of subunit uL16 in the Escherichia coli ribosome. Our work establishes an enzyme with a previously misannotated function, EcYcaO, as the catalyst responsible for thioamidation of uL16. We further assess the EcuL16-EcYcaO interaction through site-directed mutagenesis, in vitro reconstitution, and comprehensive computational modeling, unveiling an elaborate protein-protein interaction unlike anything previously observed in YcaO enzymes. We then use sequence similarity to chart a map of this modification in Nature and reconstitute two additional examples in Klebsiella pneumoniae and Pseudomonas aeruginosa. This work sets the stage for assessing both the mechanism and functional ramifications of ribosome thioamidation, subjects we are eager to continue investigating.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2024

Author dc:creator

• Rice, Andrew James

Contributors dc:contributor

• Mitchell, Douglas A
• van der Donk, Wilfred A
• Nair, Satish K
• Olshansky, Lisa

• Ripps
• Enzymes
• Natural Products
• Thiopeptide
• Halogenase
• Thioamide
• Ribosome
• Biocatalysis
• Chemical Biology

Statement dc:rights

• Copyright 2024 Andrew Rice

Language dc:language

eng, en

Handle dc:identifier

https://hdl.handle.net/2142/127354

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/127354