Investigation of radical SAM-mediated thioether installation in RiPP natural products

Precord, Timothy Wayne

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https://hdl.handle.net/2142/120339 Copy

Description

Title

Investigation of radical SAM-mediated thioether installation in RiPP natural products

Author(s)

Precord, Timothy Wayne

Issue Date

2023-02-14

Director of Research (if dissertation) or Advisor (if thesis)

Mitchell, Douglas A

Doctoral Committee Chair(s)

Mitchell, Douglas A

Committee Member(s)

• van der Donk, Wilfred A
• Hergenrother, Paul J
• Metcalf, William W

Department of Study

Chemistry

Discipline

Chemistry

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Radical SAM Enzyme
• Sactipeptide
• Thioether
• RiPPs
• Enzyme Function
• Genomic Enzymology
• Bioinformatics

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) have received considerable attention in recent years for the growing breadth of post-translational modifications (PTMs) they possess. A particularly remarkable subset of RiPPs are those that receive PTMs from radical S-adenosylmethionine (rSAM) enzymes. These chemical modifications include C-methylation, C(sp2)-C(sp3) and C(sp3)-C(sp3) crosslinking, carbon-heteroatom crosslinking, and various complex rearrangements. RiPPs containing rSAM enzyme-mediated thioether crosslinking are a growing group of natural products containing members with antimicrobial activity and the potential for engineering. The radical thioether-containing RiPPs are composed of two groups, the sulfur-to-alpha carbon thioether crosslinked peptides (sactipeptides) and the radical non-alpha thioether peptides (ranthipeptides). Chapter 1 details the chemical modifications performed by rSAM enzymes on RiPP substrates, with special emphasis placed on the sactipeptide and ranthipeptide classes. Chapter 2 elaborates on the distinction between ranthipeptides and sactipeptides through an in vivo investigation of the substrate scope of the ranthipeptide freyrasin, from Paenibacillus polymyxa. Freyrasin contains six thioether linkages between Cys and the β-carbon of Asp in subsequent Cys-X3-Asp motifs (where X is any amino acid residue). Unlike the sequentially-installed thioether crosslinks in most sactipeptides, the freyrasin maturase rSAM (PapB) was found to process all available crosslinking locations in spite of the absence of individual crosslinking residues. PapB was also able to crosslink a Cys to Glu variant at the γ-carbon. The chapter further discusses the dependence of PapB on an N-terminal RiPP-recognition element to properly install thioether linkages in the freyrasin precursor peptide. Finally, the activity of PapB was reconstituted in vitro, indicating its potential utility in RiPP engineering. Chapter 3 addresses the difficulty of unifying the sactisynthases into a single functional group in silico. Due to the high diversity of function within the rSAM protein family, the sactisynthases are paraphyletically distributed, precluding the researcher’s ability to generate a dataset of all sactisynthases with ease. Such a distribution also suggests that additional sactisynthases remain undiscovered due to sequence divergence from characterized sactisynthases. Comparative analysis of residues in proximity to the catalytic site in the sactisynthases was found to discriminate them from rSAM enzymes of different function. This catalytic proximity profiling analysis was further used to discover a new sactisynthase from Streptomyces sparsogenes, despite its high sequence similarity to the rSAM enzyme involved in mycofactocin biosynthesis, which performs oxidative decarboxylation followed by carbon-carbon crosslinking. The RiPP product from this pathway was termed ‘sparsactin’ and its possession of sulfur-to-alpha thioethers was confirmed by chemical degradation and isotopic labeling. Furthermore, catalytic proximity profiling was also used to predict additional new groups of sactisynthases that would have been undiscoverable by full-length sequence analysis. These studies were in part facilitated by the development of RadicalSAM.org, a web resource that vastly expands the bioinformatician’s toolkit for in silico rSAM protein analysis. RadicalSAM.org is detailed in Appendix A.

Graduation Semester

2023-05

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/120339

Copyright and License Information

Copyright 2023 Timothy Precord

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