A crucial enzyme in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous D-glucose 6-phosphate (G-6-P) into the specific carbocycle 2-deoxy-scyllo-inosose. Among all the oxygenated carbons of the substrate, C-1, -4, -5, and -6 are directly involved in the chemical transformation. To get insight into the roles of C-2 and C-3 hydroxy groups, 2-deoxy-2-fluoro-, 3-deoxy-3-fluoro-, 2-amino-2-deoxy-, and 3-amino-3-deoxy-D-glucose 6-phosphates (2-F-G-6-P, 3-F-G-6-P, 2-NH(2)-G-6-P, and 3-NH(2)-G-6-P, respectively) were subjected to the DOIS reaction as probe, since a fluorine substituent generally acts as a hydrogen-bond acceptor, and an ammonium functionality derived physiologically from an amino group as a hydrogen-bond donor. Among those tested, 2-F-G-6-P and 3-NH(2)-G-6-P were used as substrates by DOIS and were converted into the corresponding deoxyfluoro- and aminodeoxy-scyllo-inososes, respectively. In contrast, 3-F-G-6-P and 2-NH(2)-G-6-P were inactive in the cyclization reaction. Clearly, DOIS recognizes the G-6-P substrate through specific hydrogen-bonding interactions, i.e., through a hydrogen-donating group for C-2 and an accepting group for C-3 of the substrate. Modeling of DOIS based on the structure of evolutionary-related dehydroquinate synthase is also described.