Protein Thioether Crosslinks Installed by a SPASM-Containing Radical SAM Enzyme from Methanogenic Archaea

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.