Amide synthases catalyze the formation of macrolactam rings from aniline-containing polyketide-derived seco-acids as found in the important class of ansamycin antibiotics. One of these amide synthases is the geldanamycin amide synthase GdmF, which we recombinantly expressed, purified and studied in detail both functionally as well as structurally. Here we show that purified GdmF catalyzes the amide formation using synthetically derived substrates. The atomic structures of the ligand-free enzyme and in complex with simplified substrates reveal distinct structural features of the substrate binding site and a putative role of the flexible interdomain region for the catalysis reaction.