Linaridin is a small class of peptide natural products belonging to the ribosomally synthesized and post-translationally modified peptides (RiPPs) superfamily. By an extensive genome-wide survey of linaridin biosynthetic genes, we show that this class of natural products is widespread in nature and possesses vast structural diversity. The linaridin precursor peptides are relatively conserved in the N-termini but have diverse sequences in the core region, which appear to have coevolved with the biosynthetic enzymes. Using the prototypic linaridin cypemycin as a model, we have explored the structure-activity relationships involved in precursor peptide maturation and generated a diverse set of novel cypemycin variants, among which the T2S variant exhibits enhanced activity against Micrococcus luteus. Our results reveal valuable insights into linaridin biosynthesis and highlight the potential to explore this class of natural products by genome mining and by biosynthetic engineering studies.

S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an S 2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5'-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5'-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.

HemN is a radical S‐adenosyl‐l‐methionine (SAM) enzyme that catalyzes the oxidative decarboxylation of coproporphyrinogen III to produce protoporphyrinogen IX, an intermediate in heme biosynthesis. HemN binds two SAM molecules in the active site, but how these two SAMs are utilized for the sequential decarboxylation of the two propionate groups of coproporphyrinogen III remains largely elusive. Provided here is evidence showing that in HemN catalysis a SAM serves as a hydrogen relay which mediates a radical‐based hydrogen transfer from the propionate to the 5′‐deoxyadenosyl (dAdo) radical generated from another SAM in the active site. Also observed was an unexpected shunt product resulting from trapping of the SAM‐based methylene radical by the vinyl moiety of the mono‐decarboxylated intermediate, harderoporphyrinogen. These results suggest a major revision of the HemN mechanism and reveal a new paradigm of the radical‐mediated hydrogen transfer in radical SAM enzymology.

Sactionine-containing antibiotics (sactibiotics) are ag rowing class of peptide antibiotics belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily.W er eport the characterization of thuricin Z, an ovel sactibiotic from Bacillus thuringiensis. Unusually,t he biosynthesis of thuricin Zi nvolves two radical S-adenosylmethionine (SAM) enzymes,T hzC and ThzD. Although ThzC and ThzD are highly divergent from each other,these two enzymes produced the same sactionine ring in the precursor peptide ThzA in vitro.T huricin Ze xhibits narrow-spectrum antibacterial activity against Bacillus cereus.Aseries of analyses,i ncluding confocal laser scanning microscopy, ultrathin-sectioning transmission electron microscopy, scanning electron microscopy, and large-unilamellarvesicle-based fluorescence analysis,s uggested that thuricin Z binds to the bacterial cell membrane and leads to membrane permeabilization.

Nosiheptide is a prototypal thiopeptide antibiotic, containing an indole side ring in addition to its thiopeptide-characteristic macrocylic scaffold. This indole ring is derived from 3-methyl-2-indolic acid (MIA), a product of the radical S-adenosylmethionine enzyme NosL, but how MIA is incorporated into nosiheptide biosynthesis remains to be investigated. Here we report functional dissection of a series of enzymes involved in nosiheptide biosynthesis. We show NosI activates MIA and transfers it to the phosphopantetheinyl arm of a carrier protein NosJ. NosN then acts on the NosJ-bound MIA and installs a methyl group on the indole C4, and the resulting dimethylindolyl moiety is released from NosJ by a hydrolase-like enzyme NosK. Surface plasmon resonance analysis show that the molecular complex of NosJ with NosN is much more stable than those with other enzymes, revealing an elegant biosynthetic strategy in which the reaction flux is controlled by protein–protein interactions with different binding affinities.

S‐[(Z)‐2‐aminovinyl]‐d‐cysteine (AviCys) is a unique motif found in several classes of ribosomally synthesized and post‐translationally modified peptides (RiPPs). Biosynthesis of AviCys requires flavin‐dependent Cys decarboxylases, which are highly divergent among different RiPP classes. In this study, we solved the crystal structure of the cypemycin decarboxylase CypD. We show that CypD is structurally highly similar to lanthipeptide decarboxylases despite the absence of sequence similarities between them. We further show that Cys decarboxylases from four RiPP classes have evolved independently and form two major clusters. These results reveal the convergent evolution of AviCys biosynthesis and suggest that all the flavin‐dependent Cys decarboxylases likely have a similar Rossmann fold despite their sequence divergences.

The cypemycin decarboxylase CypD is investigated by using a synthetic oligopeptide, which contains the to-becyclized dehydroalanine (Dha) residue. It was shown that CypD efficiently catalyzes the decarboxylation of this Dha-containing peptide, but the expected AviCys ring is not formed in the product, suggesting that CypD alone is not enough to form the AviCys ring. It was also shown that the Dha-containing peptide is a better substrate than two similar peptides with a Ser or a Cys residue, supporting that, in cypemycin biosynthesis, Dha formation is prior to decarboxylation of the C-terminal Cys.

Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide (RiPP) superfamily. In this study, a genome mining approach led to the identification of a novel linaridin, mononaridin (MON), from Streptomyces monomycini. In-frame deletion genetic knockout studies showed that, in addition to many genes essential for MON biosynthesis, monM encodes an S-adenosyl methionine (SAM)-dependent α-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus. Besides SAM, MonM also accepts ethyl-SAM and allyl-SAM, in which the methyl of SAM is replaced by an ethyl and an allyl, respectively. We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis, and this observation was further investigated in detail by density functional theory (DFT) calculations. Remarkably, MonM acts efficiently on nisin, a prototypic lantibiotic that is structurally very different from the native substrate, and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin, which can be further used in microscopic cell analysis. Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.