Biosynthetic and bioinformatic investigation of RiPPs with radical SAM-installed crosslinks

Ribosomally synthesized and post translationally modified peptides (RiPPs) are an expanding class of natural products containing new chemical modalities and bioactive molecules. Although with diverse biological functions, significant research has been focused on investigating RiPPs with antibacterial activity due to the emerging antimicrobial resistance crisis. Radical S-adenosylmethionine enzymes (rSAM) are responsible for producing intriguing and complex molecules, some of which are antibiotics. These enzymes have been demonstrated to catalyze C-C, C-S, C-O-C, and C-N crosslink formation among other fascinating modifications. However, there is still work to be done in this area to further understand these enzymes and their products. In Chapter 1, I review RiPPs and rSAM modifications. Three recently described gram-negative selective antibiotics darobactin A, dynobactin A, and xenorceptide A2 have increased the prominence of cyclophane-containing rSAM-modified RiPPs. These molecules each target the β-barrel assembly machinery complex (BAM) in gram-negative bacteria. Chapter 2 focuses on the biosynthesis of darobactin A, combining experimental and computational analyses to investigate crosslink differentiation. Darobactin A, a heptapeptide, is modified by the rSAM DarE to contain a fused ether and C-C crosslinks. Despite significant work in the field, there had been little investigation on DarE with respect to substrate scope and differential ether vs C-C crosslink formation. Therefore, we investigated the enzymatic tolerance of DarE towards the modified residues of DarA and produced 51 modified variants, 10 with a bicyclic structure. We determined that DarE is under substrate control as an aromatic residue in position 3 of a crosslinking motif will dictate ether formation, while a non-aromatic residue will result in a C-C crosslink. This was further reinforced with quantum mechanical modeling and docking to provide a rationale for crosslink differentiation. The production of two novel darobactins led us to generate guidelines to consider when producing future darobactin derivatives. Our work provides the baseline for darobactin-type engineering and further elucidates rSAM catalysis. Darobactin, while one of the most prominent members, is not the sole rSAM cyclophane-containing RiPP. Founded with the structural and enzymatic elucidation of streptide, and a pyrroloquinoline quinone biosynthetic intermediate, this class has expanded greatly since. However, previous bioinformatic analyses have been limited, requiring extensive manual curation to identify cyclophane precursors. In Chapter 3 I discuss the generation of a RODEO module (a genome mining tool) to enable automatic scoring and classification of rSAM cyclophane RiPP precursor peptides. Focusing on enzyme- and precursor-centric analyses, we identify >20,000 potential rSAM cyclophane precursors, a significant increase over previous work. Bioinformatic analysis of the precursor dataset indicates previously unseen cyclophane BGC architecture with unusual precursor peptides. A large swath of our dataset contains minimally characterized radical SAM cyclophane forming enzymes (rCFEs), providing a roadmap to new cyclophane discovery. This study bioinformatically uncovers new rSAM cyclophane RiPPs, greatly expanding the class and providing extensive research opportunities to experimentally characterize these new molecular structures and potential bioactivities. While the focus of the previous chapters is on cyclophane-containing RiPPs with C-C and C-heteroatom crosslinks, there is also an emerging modality in RiPP-like large proteins modified by RiPP-associated enzymes. In Chapter 4, we investigate a large cystine rich protein (CrpA; >10% cysteine content) that is modified by an rSAM enzyme. Bioinformatic analysis of prokaryotes identified that methanogenic archaea have unusually high proteomic cysteine content, leading us to investigate cysteine-rich proteins in their proteomes and discovering the Crp biosynthetic gene cluster (BGC). In vitro experimentation demonstrated that CprC is an rSAM enzyme with three [4Fe-4S] clusters (including a SPASM domain with additional [4Fe-4S] clusters), while mass spectrometry analyses determined that CrpC installs thioether crosslinks onto CrpA These results provide another example of RiPP-like modifications on large proteins and further probes archaeal RiPP pathways.

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

• Woodard, Austin Michael

Contributors dc:contributor

• Mitchell, Douglas A
• Silverman, Scott K
• Manesis, Anastasia C
• Mehta, Angad P

• Chemical Biology
• Biochemistry
• Bioinformatics
• RiPPs
• Radical SAM enzymes
• Darobactin
• Cyclophane natural products

Statement dc:rights

• Copyright 2025 Austin Woodard

Language dc:language

en

Handle dc:identifier

https://hdl.handle.net/2142/132771

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/132771