How a Subfamily of Radical S-Adenosylmethionine Enzymes Became a Mainstay of Ribosomally Synthesized and Post-translationally Modified Peptide Discovery

Abstract

Radical S-adenosylmethionine (rSAM) enzymes are a large and diverse superfamily of enzymes, some of which are known to participate in the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs). Specifically, a subfamily of rSAM proteins with an elongated C-terminus known as a SPASM domain have become a fixation in the discovery of new RiPP natural products. Arguably, a structural study, a bioinformatic study, and a functional study built the foundation of the research for rSAM-SPASM-protein-modified RiPPs. In this Review, we focus on these three studies and how they initiated what has become an increasingly productive field. In addition, we discuss the current state of RiPPs that depends on rSAM-SPASM proteins and provide guidelines to consider in future research. Lastly, we discuss how genome mining tools have become a powerful means to identify and predict new RiPP natural products. Despite the state of our current knowledge, we do not completely understand the relationship of rSAM-SPASM chemistry, substrate recognition, and the structure–function relationship as it pertains to RiPP biosynthesis, and as such, there remain many interesting findings waiting to be discovered in the future.

The Biosynthetic Landscape of Triceptides Reveals Radical SAM Enzymes That Catalyze Cyclophane Formation on Tyr- and His-Containing Motifs

“…Our functional studies allow further comparison among the three organism sources of three-residue cyclophanes containing a single aromatic ring (Figure ). All bacterial-derived rSAM enzymes catalyze cyclophane formation via a C–C bond . The exception is DarE, which catalyzes both C–C and ether cross-links .…”

“…The cyclization reactions occur strictly via a C­(sp 2 )–Cβ­(sp 3 ) cross-link between the aromatic ring of Ω1 and Cβ of X3 in Ω1-X2-X3 motifs (Ω1 = Trp, Phe, Tyr, or His). The enzymes described here add to the growing number of rSAM enzymes able to catalyze unique cyclization reactions . Our bioinformatic analysis demonstrates that triceptides are among the larger RiPP families that have so far remained cryptic and are a resource for biocatalysts and natural product discovery.…”

“…All bacterial-derived rSAM enzymes catalyze cyclophane formation via a C−C bond. 56 The exception is DarE, which catalyzes both C−C and ether cross-links. 3 The unique ether linkage is suggested as a signature of DarE-type rSAM enzymes.…”

“…The enzymes described here add to the growing number of rSAM enzymes able to catalyze unique cyclization reactions. 56 Our bioinformatic analysis demonstrates that triceptides are among the larger RiPP families that have so far remained cryptic and are a resource for biocatalysts and natural product discovery. Genome mining in this structural class represents a unique approach.…”

Synthesis of Amino Acid α-Thioethers and Late-Stage Incorporation into Peptides

“…Subtilosin A was the first sactipeptide to be discovered back in 1985, although its structure remained elusive until pioneering work by the Vederas group in 2003 uncovered the unusual cross-linked structurecomprising three sactionine linkagesusing detailed NMR studies . Advances primarily in genome mining techniques have since fueled the rapid expansion of the sactipeptide class, which now includes, among others, several natural products from Bacillus sp. (thurincin H, sporulation killing factor (SKF), thuricin CD, thuricin Z , ), ruminococcin C produced by Ruminococcus gnavus and streptosactin produced by Streptococcus thermophilus.…”