AbstractIn the biosynthesis of the lincosamide antibiotic celesticetin, the condensation enzyme CcbD generates the lincosamide pharmacophore by forming an amide bond between the carrier protein (CP)-tethered proline and ergothioneine-conjugated thiooctose. Although the function of CcbD has been investigated, its substrate specificity, structures and catalytic mechanisms remain unclear. Here we show the structure–function analyses of CcbD. Our biochemical analysis revealed that CcbD exhibits promiscuous substrate specificity towards CP-tethered acyl substrates to generate unnatural lincosamides. Furthermore, structural analyses indicated that CcbD possesses an unusual overall fold, while the N-terminal region shows weak similarity to cysteine proteases. Thus, CcbD, like cysteine proteases, utilizes the Cys-His-Glu catalytic triad to form amide bonds in a CP-dependent manner, which is significantly different from other known amide bond-forming enzymes. Furthermore, the structures of the CcbD/thiooctose complex and the cross-linked CcbD/CcbZ-CP complex, as well as structure-based mutagenesis, revealed the intimate structural details of the CP-dependent amide bond formation reaction.