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•) 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.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.