biostudies-literatureGuo SNational Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund2361https://www.ebi.ac.uk/biostudies/studies/S-EPMC9055067biostudies-literatureUnknown13(1)Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C-C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H₂¹⁸O, apparent ¹⁸O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process inNature communicationsRadical SAM-dependent ether crosslink in daropeptide biosynthesis.PMC90550672018Y F A0900402Ding WMa SWang SDeng ZGuo SZhang QfalseRadical SAM-dependent ether crosslink in daropeptide biosynthesis.Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C-C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H₂¹⁸O, apparent ¹⁸O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in2022-01-01T00:00:00Z2022 Apr2025-04-05T13:23:25.844Z2025-04-05T13:23:25.844ZS-EPMC90550673548792110.1038/s41467-022-30084-2