Enzymatic Carbocycle Formation in Microbial Secondary Metabolism. The Mechanism of the 2-Deoxy-scyllo-inosose Synthase Reaction as a Crucial Step in the 2-Deoxystreptamine Biosynthesis in Streptomyces fradiae

The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of a major 2-deoxystreptamine aglycon of clinically important aminoglycoside antibiotics, was investigated with a partially purified enzyme fraction from neomycin-producing Streptomyces fradiae IFO 13147. Singly and doubly labeled d-[4-²H]- and D-[4-²H,3-¹⁸O]glucose-6-phosphate were used for chase experiments, and the 2-deoxy-scyllo-inosose product was analyzed by ²H-NMR and GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the product without scrambling the doubly-labeled isotopes. Since the oxidative process with the aid of NAD⁺ is essential, which was reported previously, the hydride abstraction and returning appear to take place within the same glucose molecule. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-seyllo-inosose synthase enzyme with a catalytic requirement of NAD cofactor, the mechanism of which is closely resembled to the dehydroquinate synthase in the shikimate pathway.