Radical SAM Enzymes in the Biosynthesis of Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs)

Abstract

Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large and diverse family of natural products. They possess interesting biological properties such as antibiotic or anticancer activities, making them attractive for therapeutic applications. In contrast to polyketides and non-ribosomal peptides, RiPPs derive from ribosomal peptides and are post-translationally modified by diverse enzyme families. Among them, the emerging superfamily of radical SAM enzymes has been shown to play a major role. These enzymes catalyze the formation of a wide range of post-translational modifications some of them having no counterparts in living systems or synthetic chemistry. The investigation of radical SAM enzymes has not only illuminated unprecedented strategies used by living systems to tailor peptides into complex natural products but has also allowed to uncover novel RiPP families. In this review, we summarize the current knowledge on radical SAM enzymes catalyzing RiPP post-translational modifications and discuss their mechanisms and growing importance notably in the context of the human microbiota.

Keywords: RiPPs; enzyme mechanism; iron sulfur; iron-sulfur proteins; natural products; radical AdoMet; radical SAM; ribosomally synthesized and post-translationally modified peptides.

Figure 1

(A) General mechanism of radical SAM enzymes. The radical SAM [4Fe-4S] clusters interacts with S-adenosyl-L-methionine (SAM). After the reductive SAM cleavage, the 5′-dA radical species is formed and generally abstracts a substrate H-atom. A radical substrate intermediate is formed which, after subsequent rearrangements, lead to the product. In most of the cases, SAM is used as a co-substrate (right pathway). However, several enzymes recycle SAM during catalysis (left pathway). (B) Structure of a radical SAM enzyme (Spore photoproduct lyase, PDB:4FHD, Benjdia et al., 2012) showing the radical SAM [4Fe-4S] center (in orange and yellow spheres) in interaction with the SAM cofactor.

Figure 2

Structures of RiPPs post-translationally modified by radical SAM enzymes. In red are highlighted the modifications catalyzed by radical SAM enzymes. Subtilosin A (thioether bonds), bottromycin A (methylations), thiostrepton A (methylation) (the quinaldic acid moiety is indicated by red dashed lines), epipeptide (epimerization), and polytheonamide A (epimerization and methylations).

Figure 3

Proposed mechanisms for class B (B₁₂-dependent) and class C radical SAM enzymes. (A) General architecture of B₁₂-dependent radical SAM enzymes. (B) Proposed mechanism for TsrM, an sp2-hybridized carbon methyltransferase. (C) Proposed mechanism for PoyC, an sp3-hybridized carbon methyltransferase. In contrast to TsrM, PoyC catalyzes SAM homolytic cleavage likely to abstract a substrate C_β H-atom. This intermediate is likely to react with a methyl radical species leading to the formation of C_β methyl-valine. In both mechanisms, Cob(II)alamin is likely formed. After further reduction, Cob(I)alamin can react with SAM to regenerate methyl-cobalamin for the next catalytic cycle. (D) Proposed mechanism for NosN, a class C methyltransferase. NosN uses two molecules of SAM to generate 5′-dA radical and 5′-methylthioadenosine (MTA). The 5′-dA radical abstracts an H-atom from the methyl group of MTA leading to the addition of the radical intermediate to the C-4 of the indolyl substrate. After C-S bond cleavage, 5′-thioadenosine (5′-TdA) and the methylated indole product are released.

Figure 4

Thioether bond formation by radical SAM enzymes. (A) Sequence alignment of AlbA and anSME showing the cysteine residues involved in the coordination of the [4Fe-4S] clusters present in the SPASM-domain. Cysteine residues involved in the auxiliary cluster I (Aux I) and auxiliary cluster II (Aux II) are indicated in blue and red, respectively. Numbers indicate the respective positions of the cysteines in the protein sequences of AlbA and anSME. (B) Proposed mechanism for AlbA. AlbA catalyzes H-atom abstraction on C_α-atom. The carbon-centered radical rearranges leading to the formation of an N-acyliminium intermediate. This intermediate is quenched by the thiolate group of a cysteine residue resulting in the formation of a C_α-thioether bond. The auxiliary clusters I and II are proposed to serve as an electron conduit.

Figure 5

Proposed mechanism for YydG, a radical SAM epimerase. Similarly to AlbA (Figure 4B), YydG catalyzes C_α H-atom abstraction leading to the loss of the amino acid stereochemistry. An enzyme cysteine residue (Cys²²³) provides an H-atom to the carbon-centered radical intermediate, resulting in peptide epimerization. The additional [4Fe-4S] cluster located in the C-terminus end of the protein, likely assists the quenching of the thiyl radical formed during the reaction and regenerates the cysteine H-atom donor, for the next catalytic cycle.

Figure 6

Post-translational modifications catalyzed by Radical SAM enzymes on RiPPs.