Catalytic Site Proximity Profiling for Functional Unification of Sequence-Diverse Radical S-Adenosylmethionine Enzymes

Timothy W. Precord, Sangeetha Ramesh, Shravan R. Dommaraju, Lonnie A. Harris, Bryce L. Kille, Douglas A. Mitchell

Research output: Contribution to journalArticlepeer-review

Abstract

The radical S-adenosylmethionine (rSAM) superfamily has become a wellspring for discovering new enzyme chemistry, especially regarding ribosomally synthesized and post-translationally modified peptides (RiPPs). Here, we report a compendium of nearly 15,000 rSAM proteins with high-confidence involvement in RiPP biosynthesis. While recent bioinformatics advances have unveiled the broad sequence space covered by rSAM proteins, the significant challenge of functional annotation remains unsolved. Through a combination of sequence analysis and protein structural predictions, we identified a set of catalytic site proximity residues with functional predictive power, especially among the diverse rSAM proteins that form sulfur-to-α carbon thioether (sactionine) linkages. As a case study, we report that an rSAM protein from Streptomyces sparsogenes (StsB) shares higher full-length similarity with MftC (mycofactocin biosynthesis) than any other characterized enzyme. However, a comparative analysis of StsB to known rSAM proteins using “catalytic site proximity” predicted that StsB would be distinct from MftC and instead form sactionine bonds. The prediction was confirmed by mass spectrometry, targeted mutagenesis, and chemical degradation. We further used “catalytic site proximity” analysis to identify six new sactipeptide groups undetectable by traditional genome-mining strategies. Additional catalytic site proximity profiling of cyclophane-forming rSAM proteins suggests that this approach will be more broadly applicable and enhance, if not outright correct, protein functional predictions based on traditional genomic enzymology principles.

Original languageEnglish (US)
Pages (from-to)240-251
Number of pages12
JournalACS Bio and Med Chem Au
Volume3
Issue number3
DOIs
StatePublished - Jun 21 2023

Keywords

  • RiPP
  • bioinformatics
  • enzyme function
  • genomic enzymology
  • radical SAM enzyme
  • sactipeptide
  • thioether

ASJC Scopus subject areas

  • Drug Discovery
  • Molecular Biology
  • Biochemistry
  • Pharmaceutical Science

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