# Enzyme Chemistry and Biological Function of RiPP-like Modifications

> **NIH NIH R01** · UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN · 2024 · $1

## Abstract

Our group seeks to identify new molecular structures formed by unusual enzymatic transformations. We focus
on the ribosomally synthesized and post-translationally modified peptides (RiPPs), of which nearly 50 distinct
structural classes exist. Decades of research show that while RiPPs harbor diverse structures and functions,
the biosynthetic routes share a blueprint. Typical RiPP precursor peptides contain fewer than 60 amino acids.
The modification enzymes engage the N-terminal portion, while the C-terminal region receives all post-trans-
lational modifications (PTMs). Genome mining for RiPP biosynthetic gene clusters (BGCs) had been ex-
tremely time-consuming and often unsuccessful owing to the difficulty of locating the requisite substrate pep-
tide(s). The short length and hypervariability of RiPP precursor peptides frequently preclude their detection
by automated gene finders. RODEO, an AI-based genome mining tool, has largely solved this problem for a
subset of RiPP classes. The current proposal unites RODEO-enabled genomics analysis, enzyme chemistry,
structural biology, and microbial physiology to characterize several “RiPP-like” BGCs that do not conform to
the current definition of a RiPP natural product.
With the breadth and depth of RiPP genomics coming into sharper focus, it has become increasingly clear
that many “RiPP-like” BGCs lack a canonical precursor peptide. Rare PTMs such as backbone thioamidation
and thioether crosslinks between cysteine and the side chains of other amino acids are known on larger
protein substrates, such as methyl-coenzyme M reductase, ribosomal protein uL16, and quinohemoprotein
amine dehydrogenase. These examples establish limited but critical precedent, and we herein predict that
many more “RiPP-like PTMs” occur on protein substrates. Comparative analysis in prokaryotes supports this
prediction, as a substantial level of sequence and genome neighborhood similarity exists between pathways
encoding a canonical precursor peptide versus a larger protein substrate.
This renewal project tackles several questions of outstanding interest regarding the interplay and utility of
RiPP-like PTMs on non-canonical substrates. The specific aims are independently achievable and robustly
combine in silico, in vitro, and in vivo methods. In one aspect, we will elucidate the biochemical function of
ribosome thioamidation and the extent to which this PTM is propagated among other prokaryotes. Additional
thioamidated proteins and their physiological roles will be discovered during this project. Further, we will pur-
sue biosynthetic pathways predicted to produce poly-thioether-stabilized protein nanotubes and poly-isopep-
tide-containing branched copolymers. The enzymes performing this usual PTM chemistry will be thoroughly
evaluated, and the biological roles of the protein ultrastructures will be determined. Our preliminary data, rich
environment, and strong investigative team place us in an ideal position to address these a...

## Key facts

- **NIH application ID:** 10881537
- **Project number:** 2R01GM097142-13
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
- **Principal Investigator:** Douglas Alan Mitchell
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $1
- **Award type:** 2
- **Project period:** 2012-02-01 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10881537

## Citation

> US National Institutes of Health, RePORTER application 10881537, Enzyme Chemistry and Biological Function of RiPP-like Modifications (2R01GM097142-13). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10881537. Licensed CC0.

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