Function, timing and catalytic mechanism of NosN, a class C radical SAM methylase involved in the biosynthesis of nosiheptide's side‐ring system

Abstract

Nosiheptide is a bicyclic thiopeptide natural product that exhibits potent antibiotic activity against a number of clinically important Gram‐positive pathogens. It is biosynthesized from a ribosomally produced core peptide that is then modified extensively to contain a number of thiazole rings and dehydrated amino acids, as well as other modifications. It also contains a unique side‐ring structure composed of a 3,4‐dimethylindolic acid bridge connected to the side chains of Glu6 and Cys8 of the core peptide via ester and thioester linkages, respectively. In addition to the core peptide, encoded by the nosM gene, the biosynthesis of the side‐ring structure requires the actions of NosI, J, K, L, and N. NosN is annotated as a class C radical S‐adenosylmethionine (SAM) methylase, but little is known about how the enzyme functions or what its true substrate is. By designing, synthesizing and testing small peptides as substrate mimics, we show that the true function of NosN is to transfer a C1 unit from SAM to C4 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. In addition, using full‐length peptides, we show that NosN completes installation of the side‐ring structure immediately after NosK appends MIA to the core peptide. Using a substrate mimic containing a rigid structure, we also identify and characterize two reaction‐based adducts containing SAM fused to C4 of MIA The two SAM‐adducts are derived from a consensus radical‐containing species proposed to be the key intermediate in our proposed catalytic mechanism of NosN.Support or Funding InformationThis work was supported by NIH (GM‐122595 and AI‐133318 to S.J.B.). S.J.B. is an investigator of the Howard Hughes Medical Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.