Fig. 1: Chemical structures of STs and ST-related antibiotics.

a The gene product of orf1 encoded in the BD-12 producing strain is able to catalyze glycinothricin to BD-12. Other than glycinothricin, glycylthricin, 3-aminopropionylthricin, 4-aminobutylthricin and ST-F can serve as substrates of Orf1 to bring on N-formimidoylated or N-iminoacetylated corresponding products. b The N-formimidoylation or N-iminoacetatylation was presumed to follow multiple steps of reactions in one single reaction site as does a typical FAD-dependent enzyme. c The proposed mechanisms of N-formimidoylation or N-iminoacetylation catalyzed by Orf1 that recruits a FAD prosthetic group and an NOS protein-ligand covalent modification to enable multiple reactions to take place at two separate but adjacent reaction chambers. Four-membered peroxide and three-membered oxaziridine that may respectively react with oxidized and non-oxidized cysteine to form the NOS linkage, however, were not detected in any circumstances. In the presence of proper acceptors (e.g., 1 or 4), products 2, 5 with the N-formimidoyl modification are formed by Orf1, in which sulfenic C281 is recyclable (reactivated only once). R’HN’ colored blue is glycylthricin analogs (e.g., compound 7) with various aliphatic side chains in length leading to N-iminoacetyl modification. In this circumstance, apart from hydrogen peroxide (H₂O₂) sulfenic C281 can be regenerated by water as determined by isotope labeling experiments using H₂¹⁸O highlighted red (see Fig. 5c).