2026-10-10T22:03:32Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33949415 2026-09-21T05:04:17Z category_1 category_4 category_45 category_873 category_502 category_734 category_69 category_132 portal_63 item_type_3 month_year_09_2026

Protein Thioether Crosslinks Installed by a SPASM-Containing Radical SAM Enzyme from Methanogenic Archaea Emily M. Dieter (25080107) Austin M. Woodard (25080110) Jared C. Green (25080113) Jin Xiong (3359327) Yisong Guo (1549507) William E. Broderick (2144131) Douglas A. Mitchell (1314975) Joan B. Broderick (1336659) Biophysics Biochemistry Sociology Chemical Sciences not elsewhere classified Astronomical and Space Sciences not elsewhere classified Biological Sciences not elsewhere classified Inorganic Chemistry Infectious Diseases wide biological utility vital amino acid termed ribosomally synthesized targeted proteome scanning native biological function form thioether bridges complete structural elucidation protein binds three rich prokaryotic proteomes rich protein substrate translationally modified peptides like modifications might enzyme assays determined methanogenic archaea cysteine large protein substrates modified crpa indicates protein structures rich proteins short peptides modified cysteine translational modifications suitable candidates serving functions new path natural products methanococcus voltae mass spectrometry ligand binding investigation due intriguing bgcs experimental validation enzymes predicted enzymatic modification chemical derivatization catalyze ripp annotated radical >- adenosyl 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 <i>S</i>-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. 2026-09-21T00:00:00Z Dataset Dataset 10.1021/acsbiomedchemau.6c00133.s002 https://figshare.com/articles/dataset/Protein_Thioether_Crosslinks_Installed_by_a_SPASM-Containing_Radical_SAM_Enzyme_from_Methanogenic_Archaea/33949415 CC BY-NC 4.0