Characterizing peptide-modifying enzyme activity in RiPP biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) use enzymatic chemistry to diversify simple peptide backbones into architecturally complex and functionally rich natural products. These dedicated peptide-modifying enzymes install heterocycles, macrocycles, acyl groups, halogens, and other moieties to endow biological functions and provide their producing organisms an evolutionary advantage. This thesis focuses on mechanistic and functional studies of such enzymes. The first chapter examines ChlH, a flavin-dependent halogenase from the chlorolassin lasso peptide biosynthetic pathway. ChlH chlorinates tryptophan residues at internal and terminal positions of an assortment of tested peptide substrates. Systematic mutagenesis of its native substrate peptide reveals a high level of inherent enzyme promiscuity. This promiscuous activity was further leveraged to demonstrate halogenation capability on various non-native RiPP precursor peptides, pharmacologically relevant peptides, and even on a folded protein. Molecular dynamics simulations support the role of a C-terminal motif in substrate engagement, and lay the groundwork for future enzyme engineering campaigns. Together, these data establish ChlH as a useful biocatalyst for peptidic tryptophan halogenation. The second primary chapter characterizes a large family of multinuclear non-heme iron-dependent oxidative (MNIO) enzymes. Significant effort has been directed in recent years toward the characterization of these enzymes owing to their breadth of unique chemical reactivity. Through genome mining, members of this enzyme family were mapped and a representative gene cluster from Fontimonas thermophila was identified. By production and spectroscopic analysis of the product of this cluster, fontiphorin, MNIO-catalyzed installation of seven 5-thiooxazole (5TO) moieties was revealed. During this work, additional MNIO products were reported in the literature with conflicting structural assignments. Using alkylation-assisted HMBC correlations, it was demonstrated that these products also contain 5TO resulting in a revision of these previously reported structures. The role of 5TO-containing peptides in copper detoxification was investigated and this emerging class of Cu-associated peptidic thiooxazole metallophores was proposed to be unified under the umbrella term of captophorins. To further explore the captophorins, in vitro reconstitution of their biosynthetic machinery was performed. Using cell-free production of single-site, double-site, and naturally occurring substrate variants, enzyme-substrate interactions were examined to determine key sites governing catalysis. Three additional chapters showcase my supporting contributions towards the study of other peptide-modifying enzymes. In one, the interactions between a RiPP precursor recognition element (RRE) and its cognate lasso peptide leader peptidase was deciphered by evolutionary covariance, NMR spectroscopy, and enzyme activity assays. It was shown that the RRE both delivers the leader peptide and completes the S2 pocket of the protease, providing a new paradigm for protease regulation in RiPP pathways. In another contribution, 2D NMR spectroscopy was used to perform stereochemical assignments of a novel RiPP/fatty acid hybrid molecule. Finally, a third contribution was made towards the study of aminoacyl-tRNA specificity in a peptide aminoacyl-tRNA ligase (PEARL) enzyme. Using flexizyme-generated aminoacyl-tRNAs, both the amino acid and tRNA body were shown to jointly encode specificity while still permitting incorporation of noncanonical amino, hydroxy, and thiocarboxylic acids.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Gadgil, Mayuresh Girish

Contributors dc:contributor

• Mitchell, Douglas A
• Nair, Satish K
• Chan, Jeff
• Hergenrother, Paul J

• RiPPs
• peptides
• enzymes
• natural products
• post-translational modifications

Statement dc:rights

• Copyright 2025 Mayuresh Gadgil

Language dc:language

en

Handle dc:identifier

https://hdl.handle.net/2142/132531

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/132531