Nosiheptide,a parent compound of thiopeptide antibiotics family that exhibit potent activity against various bacterial pathogens,bears a unique indole side ring system and region specific hydroxyl groups on the characteristic macrocyclic core.In its biosynthesis,NosA catalyzes its final C-terminal amide formation by removing acrylate unit,the mechanism of which is novel,but not clear.We here report the crystal structure of the truncated inactive NosA 1-111 variant at the resolution of 2.40.Structural analysis and biochemical characterization of the enzyme,including active site scanning,unveil that NosA functions as a dimer to interact with the substrate.The positively charged K49 in one monomer and the negative charged E101 in another monomer are critical to the catalytic reaction,as well as the C-terminal NosA 112-151 peptide.NosA 1-111 displays partial catalytic power upon mixing with NosA 112-151 peptide.NMR backbone atoms dynamic experiments,and the molecular dynamics simulation reveal that K49 and the C-terminal NosA 112-151 peptide work as two “hands” to fasten the substrate.The side-chain NH 2 of K49 forms a hydrogen-bond with 3–OH group in pyridine ring of the substrate,while the NosA 112-151 peptide clutches the substrate through hydrophobic interaction and hydrogen-bond.Finally,the mechanism about how NosA catalyzes carboxyl-terminal formation of nosiheptide is presented.