The reaction stereochemistry of 2-deoxy-scyllo-inosose (DOI) synthase (DOIS) derived from butirosin-producing Bacillus circulans, the key starter enzyme of 2-deoxystreptamine biosynthesis, was closely analyzed by use of the purified recombinant DOIS. The stereochemical pathway of the DOIS reaction is common in the aminoglycoside-producing Bacillus and Streptomyces, but is distinct from a related dehydroquinate synthase reaction in the shikimate pathway. Evolutionary relationship between these enzymes is also discussed.