Abstract
Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs). These enzymes have been shown to catalyze unconventional reactions, such as methyl transfer to electrophilic carbon atoms, sulfur to Cα atom thioether bonds, or carbon-carbon bond formation. Recently, a novel radical SAM enzyme catalyzing the formation of a lysine-tryptophan bond has been identified in Streptococcus thermophilus, and a reaction mechanism has been proposed. By combining site-directed mutagenesis, biochemical assays, and spectroscopic analyses, we show here that this enzyme, belonging to the emerging family of SPASM domain radical SAM enzymes, likely contains three [4Fe-4S] clusters. Notably, our data support that the seven conserved cysteine residues, present within the SPASM domain, are critical for enzyme activity. In addition, we uncovered the minimum substrate requirements and demonstrate that KW cyclic peptides are more widespread than anticipated, notably in pathogenic bacteria. Finally, we show a strict specificity of the enzyme for lysine and tryptophan residues and the dependence of an eight-amino acid leader peptide for activity. Altogether, our study suggests novel mechanistic links among SPASM domain radical SAM enzymes and supports the involvement of non-cysteinyl ligands in the coordination of auxiliary clusters.
Highlights
Radical S-adenosylmethionine (SAM) enzymes are emerging as a major superfamily of biological catalysts involved in the biosynthesis of the broad family of bioactive peptides called ribosomally synthesized and post-translationally modified peptides (RiPPs)
As shown (Fig. 2b), the purified enzyme exhibited absorption bands at ϳ320 and ϳ410 nm after anaerobic iron-sulfur cluster reconstitution and an A420/A280 ratio of ϳ0.3, similar to what has been reported for anaerobic sulfatase-maturating enzyme (anSME), a radical SAM enzyme containing three [4Fe-4S] clusters [15]
A search in the upstream region of these radical SAM enzymes led to the identification of small ORFs coding for putative peptides containing the KGDGW motif (Fig. 3a), whereas, in the downstream regions, we identified genes coding for a putative protease and ABC transporter, similar to the S. thermophilus operon
Summary
RiPP, ribosomally synthesized and post-translationally modified peptide; SPASM, subilitosin, PQQ, anaerobic sulfatases, and mycofactocin; SAM, S-adenosylmethionine; TOCSY, total correlation spectroscopy; HSQC, heteronuclear single quantum coherence; HMBC, heteronuclear multiple bond correlation. The nature, number, and function of these metallic centers are still not well understood [21] Investigated recently, these enzymes appear to constitute one of the largest groups in the superfamily of radical SAM enzymes. The KW_cyclase from Streptococcus thermophilus has been shown to catalyze C–C bond formation between a lysine and a tryptophan residue during the biosynthesis of a unique cyclic peptide with unknown function [8, 24]. We investigated the substrate specificity and the role of conserved cysteine residues in the KW_cyclase from S. thermophilus to gain further insights into its mechanism and the large family of SPASM domain radical SAM enzymes
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