AbstractThe potent antitumor antibiotic pactamycin is an aminocyclopentitol‐containing natural product produced by the soil bacterium Streptomyces pactum. Recent studies showed that the aminocyclopentitol unit is derived from N‐acetyl‐D‐glucosamine, which is attached to an acyl carrier protein (ACP)‐bound polyketide by a glycosyltransferase enzyme, PtmJ. Here, we report a series of post‐glycosylation modifications of the sugar moiety of the glycosylated polyketide while it is still attached to the carrier protein. In vitro reconstitution of PtmS (an AMP‐ligase), PtmI (an ACP), PtmJ, PtmN (an oxidoreductase), PtmA (an aminotransferase), and PtmB (a putative carbamoyltransferase) showed that the N‐acetyl‐D‐glucosamine moiety of the glycosylated polyketide is first oxidized by PtmN and then transaminated by PtmA to give ACP‐bound 3‐amino‐3‐deoxy‐N‐acetyl‐D‐glucosaminyl polyketide. The amino group is then coupled with carbamoyl phosphate by PtmB to give a urea functionality. We also show that PtmG is a deacetylase that hydrolyses the C‐2 N‐acetyl group to give a free amine.