<jats:title>Abstract</jats:title><jats:p>The proteins Neo‐11 and Neo‐18 encoded in the neomycin gene cluster (<jats:italic>neo</jats:italic>) of<jats:italic>Streptomyces fradiae</jats:italic>NCIMB 8233 have been characterized as glucosaminyl‐6′‐oxidase and 6′‐oxoglucosaminyl:<jats:sc>L</jats:sc>‐glutamate aminotransferase, respectively. The joint activity of Neo‐11 and Neo‐18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD‐dependent dehydrogenation followed by a pyridoxal‐5′‐phosphate‐mediated transamination. Neo‐18 is also shown to catalyze deamination at C‐6′′′ of neomycin, thus suggesting bifunctional roles of the two enzymes in the formation of both neosamine rings of neomycin. The product of the<jats:italic>btrB</jats:italic>gene, a homologue of<jats:italic>neo‐18</jats:italic>in the butirosin biosynthetic gene cluster (<jats:italic>btr</jats:italic>) in<jats:italic>Bacillus circulans</jats:italic>, exhibits the same activity as Neo‐18; this indicates that there is a similar reaction sequence in both butirosin and neomycin biosynthesis.