Nosiheptide is a ribosomally synthesized and post-translationally modified thiopeptide natural product that possesses antibacterial, anticancer, and immunosuppressive properties. It contains a bicyclic structure composed of a large macrocycle and a unique side-ring system containing a 3,4-dimethylindolic acid bridge (DMIA) connected to the side chains of Glu6 and Cys8 of the core peptide viaester and thioester linkages, respectively. In addition to the structural peptide, encoded by the nosMgene, the biosynthesis of the side-ring structure requires the actions of NosI, J, K, L, and N, which is annotated as a class C radical S-adenosylmethionine (SAM) methylase. Herein, we dissect the pathway for the formation of the DMIA bridge and the side ring system, detailing the functions of the aforementioned gene products. In particular, we show that the true function of NosN is not that of a methylase, but is to transfer a methylene unit from SAM to C4 of a Cys8 thioester adduct of 3-methyl-2-indolic acid (MIA) with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C1 unit. Using a number of synthetic peptide substrate analogs, we address the timing of NosN’s function and provide evidence for the adduct between the former methyl carbon of one SAM molecule with C4 of MIA. In our mechanistic model, one SAM molecule is reductively cleaved to generate a 5’-deoxyadenosyl 5’-radical, which abstracts a hydrogen atom from the methyl moiety of a second simultaneously bound molecule of SAM. The resulting methylene radical adds to MIA at C4, affording a highly electrophilic species upon loss of a proton and an electron. The electrophilic species is then trapped by Glu6 of the core peptide, affording the DMIA bridge and creating the side-ring system.

Biosynthesis of the side-ring system of nosiheptide: Role of NosN, a class C radical S-adenosylmethionine methylase

2019 Fall National Meeting

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