Protein Thioether Crosslinks Installed by a SPASM-Containing Radical SAM Enzyme from Methanogenic Archaea.
7 affiliations
- 1Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717, United States.
- 2Department of Chemistry and Biochemistry, Gettysburg College, Gettysburg, Pennsylvania 17325, United States.
- 3Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
- 4Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States.
- 5Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States.
- 6Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
- 7Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37232, United States.
Abstract
Cysteine is a vital amino acid with wide biological utility, serving functions in catalysis, ligand binding, and maintenance of protein structures. In a subset of natural products, termed ribosomally synthesized and post-translationally modified peptides (RiPPs), multiple subclasses are defined by the enzymatic modification of cysteine residues to form thioether bridges. However, RiPPs are short peptides and experimental validation of enzymatically installed thioether crosslinks on large protein substrates is limited. We hypothesized that cysteine-rich proteins whose genes are adjacent to those for enzymes predicted to catalyze RiPP-like modifications might be substrates for such modifications. Thus, we set out to investigate cysteine-rich prokaryotic proteomes for such examples. A bioinformatic analysis identified methanogenic archaea as suitable candidates for further investigation due to the high overall proteome cysteine content, and through targeted proteome scanning, we identified a suitable biosynthetic gene cluster (BGC; Crp) from Methanococcus voltae to characterize. Purification and reconstitution of an annotated radical S-adenosyl-l-methionine (RS) enzyme, CrpC, confirmed that the protein binds three [4Fe-4S] clusters, two of which are in a SPASM domain. Enzyme assays determined that CrpC is active after reconstitution and modifies a cysteine-rich protein substrate in the same BGC, CrpA. Mass spectrometry and chemical derivatization of modified CrpA indicates that CrpC installs ranthionine crosslinks onto CrpA. This research provides a new path for the discovery of intriguing BGCs and post-translational modifications and provides a foundation for the complete structural elucidation and assessment of the native biological function of these modified cysteine-rich proteins.
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BibTeX
@article{dieter2026protein,
title = {{Protein Thioether Crosslinks Installed by a SPASM-Containing Radical SAM Enzyme from Methanogenic Archaea.}},
author = {Emily M Dieter and Austin M Woodard and Jared C Green and Jin Xiong and Yisong Guo and William E Broderick and Douglas A Mitchell and Joan B Broderick},
journal = {ACS bio \& med chem Au},
year = {2026},
month = sep,
doi = {10.1021/acsbiomedchemau.6c00133},
url = {https://europepmc.org/article/MED/42921293},
note = {CC BY},
}
RIS
TY - JOUR
TI - Protein Thioether Crosslinks Installed by a SPASM-Containing Radical SAM Enzyme from Methanogenic Archaea.
AU - Emily M Dieter
AU - Austin M Woodard
AU - Jared C Green
AU - Jin Xiong
AU - Yisong Guo
AU - William E Broderick
AU - Douglas A Mitchell
AU - Joan B Broderick
PY - 2026
DA - 2026/09/21
JO - ACS bio & med chem Au
DO - 10.1021/acsbiomedchemau.6c00133
UR - https://europepmc.org/article/MED/42921293
AB - Cysteine is a vital amino acid with wide biological utility, serving functions in catalysis, ligand binding, and maintenance of protein structures. In a subset of natural products, termed ribosomally synthesized and post-translationally modified peptides (RiPPs), multiple subclasses are defined by the enzymatic modification of cysteine residues to form thioether bridges. However, RiPPs are short peptides and experimental validation of enzymatically installed thioether crosslinks on large protein substrates is limited. We hypothesized that cysteine-rich proteins whose genes are adjacent to those for enzymes predicted to catalyze RiPP-like modifications might be substrates for such modifications. Thus, we set out to investigate cysteine-rich prokaryotic proteomes for such examples. A bioinformatic analysis identified methanogenic archaea as suitable candidates for further investigation due to the high overall proteome cysteine content, and through targeted proteome scanning, we identified a suitable biosynthetic gene cluster (BGC; Crp) from Methanococcus voltae to characterize. Purification and reconstitution of an annotated radical S-adenosyl-l-methionine (RS) enzyme, CrpC, confirmed that the protein binds three [4Fe-4S] clusters, two of which are in a SPASM domain. Enzyme assays determined that CrpC is active after reconstitution and modifies a cysteine-rich protein substrate in the same BGC, CrpA. Mass spectrometry and chemical derivatization of modified CrpA indicates that CrpC installs ranthionine crosslinks onto CrpA. This research provides a new path for the discovery of intriguing BGCs and post-translational modifications and provides a foundation for the complete structural elucidation and assessment of the native biological function of these modified cysteine-rich proteins.
SN - 2694-2437
ER -