The last stages of assembly of the aminocoumarin antibiotics, clorobiocin and coumermycin A(1), which target the GyrB subunits of bacterial DNA gyrase, involve enzymatic transfer of the pyrrolyl-2-carbonyl acyl group from a carrier protein (CloN1/CouN1) to the 3'-OH of the noviosyl moiety of the antibiotic scaffold. The enzyme, CouN7, will catalyze both the forward and back reaction on both arms of the coumermycin scaffold. This occurs via an O-acyl-Ser(101)-CouN7 intermediate, as shown by transient labeling of the enzyme with [(14)C]acetyl-S-CouN1 as donor and by inactivating mutation of the active site, Ser(101), to Ala. The intermediacy of the pyrrolyl-2-carbonyl-O-CouN7 allows net pyrrole transfer between distinct aminocoumarin scaffolds, for example, between the descarbamoylnovobiocin scaffold and coumermycin A(1) and vice versa. CouN7 also allows shuttling of surrogate acyl groups between noviosyl-aminocoumarin scaffolds to generate new antibiotic variants.