Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

Aidin R. Balo, Alessio Caruso, Lizhi Tao, Dean J. Tantillo, Mohammad R. Seyedsayamdost, R. David Britt

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

Original languageEnglish (US)
Article numbere2101571118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number21
DOIs
StatePublished - May 25 2021

All Science Journal Classification (ASJC) codes

  • General

Keywords

  • Biochemistry | enzymology | EPR spectroscopy | bioinorganic chemistry | chemical biology

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