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

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 center dot) in addition to the organometallic omega intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp center dot 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.

Automated summary

Using electron paramagnetic resonance (EPR) spectroscopy, this study dissected the mechanism of SuiB and revealed the essential role of the auxiliary [4Fe-4S] cluster in catalysis, confirming the proposed radical electrophilic aromatic substitution (rEAS) mechanism for the formation of the Lys-Trp cross-link. The findings provide direct evidence for the involvement of the SPASM domain auxiliary cluster and support rEAS as the mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.