The doubly functional aminotransferase BtrS in the 2-deoxystreptamine (DOS) biosynthesis, in which two transaminations are involved, was characterized by a genetic as well as a chemical approach with the heterologously expressed enzyme. The gene disruption study clearly showed that BtrS is involved, in addition to the previously confirmed first transamination, in the second transamination as well. This dual function of BtrS for the DOS biosynthesis was further confirmed by the structural determination of the reverse reaction product from DOS. Enantiospecific formation of the reverse reaction product from DOS clearly showed that BtrS distinguishes the enantiotopic amino groups of DOS, but in contrast, both enantiomers of 2-deoxy-scyllo-inosose (DOI) were efficiently accepted by BtrS to give a racemic product. This unique stereochemical recognition of DOI chirality and DOS prochirality by BtrS is mechanistically explained by a specific hydrogen-bond donating force in the enzyme active site as a particular feature of this doubly functional enzyme.