Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) display a diverse range of structures and continue to expand as a natural product class. Accordingly, RiPPs exhibit a wide array of bioactivities, acting as broad and narrow spectrum growth suppressors, antidiabetics, and antinociception and anticancer agents. Because of these properties, and the complex repertoire of post-translational modifications (PTMs) that give rise to these molecules, RiPP biosynthesis has been intensely studied. RiPP biosynthesis often involves enzymes that perform unique chemistry with intriguing reaction mechanisms, which attract chemists and biochemists alike to study and re-engineer these pathways. One particular type of RiPP biosynthetic enzyme is the so-called radical S-adenosylmethionine (rSAM) enzyme, which utilizes radical-based chemistry to install several distinct PTMs. Here, we describe the rSAM enzymes characterized over the past decade that catalyze six reaction types from several RiPP biosynthetic pathways. We present the current state of mechanistic understanding and conclude with possible directions for future characterization of this enzyme family.

Figure 1

rSAM enzymes catalyze a wide variety of chemical complex transformations in RiPP biosynthesis. The variety of domain architectures contained within this family echoes the observed mechanistic diversity. Class C methyltransferases are homologous to HemN (orange) and the rSAM (green) domain. It is possible that additional enzymes may actual harbor a RiPP precursor peptide Recognition Element (RRE) domain (e.g. SkfB); however, the sequence hypervariability of the RRE may preclude bioinformatic identification by tools such as HHpred. The SPASM domain of YydG (*) features only one auxiliary [4Fe-4S] cluster. SPASM domains that harbor a single auxiliary [4Fe-4S] cluster have recently been referred to as “Twitch” domains.

Figure 2

The biosynthetic gene cluster (A) for thiostrepton (3) features TsrM, an atypical rSAM MT which methylates C2 of Trp (B). Catalysis of this reaction is mediated by a cobalamin-based cofactor. Competing radical (C) and polar (D) mechanisms are presented.

Figure 3

Structure of polytheonamide A/B. Sites of sp³ C-methylation are indicated in red while the side chains of Cα epimerized sites are shown in blue. Both reactions are mediated by rSAM enzymes.

Figure 4

The biosynthetic gene cluster for polytheonamide features three rSAM enzymes: two MTs (PoyB and PoyC) as well as an epimerase (PoyD) (A). PoyB/C catalyze the C-methylation of specific residues on the PoyA precursor peptide (B) using a cobalamin-dependent reaction mechanism (C).

Figure 5

Nosiheptide (A) biosynthesis requires two rSAM enzymes, NosL and NosN. NosL converts Trp (1) to 3-methyl-2-indolic acid (62) while NosN C-methylates the indolyl moiety of 12 (a derivative of 62) prior to its incorporation into 14 (B) through a mechanism that utilizes MTA (15) as a methyl donor (C). The indolyl moiety is shown in blue for structural clarity.

Figure 6

Structurally related thiopeptides GE2270A (23) and thiomuracin (24) are encoded by similar BGCs which both feature class C rSAM MTs (A). These enzymes C-methylate the 5 position of thiazoles on a linear intermediate (B) in a regioselective manner. Both compounds feature methylation at Thz4 while GE2270A has an additional modification at Thz6 (C).

Figure 7

Several RiPP BGCs have been identified that feature a rSAM responsible for sactionine installation (A). Structure of a sactionine linkage (B). Sactipeptides exhibit variation in the number and location of the sactionine linkages (C). * denotes sactionines only observed through in vitro reconstitution (i.e. the natural product has not been characterized). Proposed mechanism for sactionine biosynthesis (D). In contrast to the radical-based mechanism of sactionine formation, lanthionines, which feature a C_β thioether linkage, are formed through a Michael-type addition (E).

Figure 8

An alternative mechanism proposed for Tte1186-dependent thioether formation.

Figure 9

Epipeptide BGC (A). Using residues 18–49 of the precursor peptide YydF (B), the installation of up to two D-amino acids was observed (C). Proposed enzymatic mechanism (D).

Figure 10

The streptide BGC from S. thermophilus encodes one rSAM enzyme (A). Structure of streptide (B). The Lys-Trp crosslinking is formed by StrB (C). Proposed StrB mechanism (D).

Figure 11

PQQ BGC from Klebsiella pneumoniae (A). PqqE catalyzes an intramolecular crosslink in PqqA (B). Proposed PqqE mechanism (C).

Figure 12

Mycofactocin is encoded by a BGC from Mycobacterium smegmatis(A) MftA biosynthetic intermediate produced (B), decarboxylation and cross-linking reactions catalyzed by MftC (C). Proposed mechanism for MftC (D).

Figure 13

NosL catalyzed transformation of 1 to 62, 63, and 64; colored atoms show the fate of the carbon atoms during rearrangement (A). Two mechanisms have been proposed for NosL (B, C).