AbstractSulfonamide is present in many important drugs, due to its unique chemical and biological properties. In contrast, naturally occurring sulfonamides are rare, and their biosynthetic knowledge are scarce. Here we identify the biosynthetic gene cluster of sulfonamide antibiotics, altemicidin, SB-203207, and SB-203208, from Streptomyces sp. NCIMB40513. The heterologous gene expression and biochemical analyses reveal unique aminoacyl transfer reactions, including the tRNA synthetase-like enzyme SbzA-catalyzed L-isoleucine transfer and the GNAT enzyme SbzC-catalyzed β-methylphenylalanine transfer. Furthermore, we elucidate the biogenesis of 2-sulfamoylacetic acid from L-cysteine, by the collaboration of the cupin dioxygenase SbzM and the aldehyde dehydrogenase SbzJ. Remarkably, SbzM catalyzes the two-step oxidation and decarboxylation of L-cysteine, and the subsequent intramolecular amino group rearrangement leads to N-S bond formation. This detailed analysis of the aminoacyl sulfonamide antibiotics biosynthetic machineries paves the way toward investigations of sulfonamide biosynthesis and its engineering.