KINETIC ISOTOPE EFFECT AND REACTION-MECHANISM OF 2-DEOXY-SCYLLO-INOSOSE SYNTHASE DERIVED FROM BUTIROSIN-PRODUCING BACILLUS-CIRCULANS

The mechanism of 2-deoxy-scyllo-inosose synthase reaction, a carbocycl e formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol anti biotics, was investigated with a partially purified enzyme from butiro sin-producing Bacillus circulans SANK 72073. Nonlabeled and double-lab eled D-[4-H-2, 3-O-18]glucose-6-phosphate were used for cross-over exp eriment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inos ose product was analyzed by GC-MS. The deuterium label at C-4 of the s ubstrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduct ion of NAD(+) cofactor was proved to be essential in the 2-deoxy-scyll o-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isot ope effect was estimated to be k(H)/k(D) = 2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deo xy-scyllo-inosose synthase enzyme with catalytic requirement of NAD(+) , the mechanism of which appears to be resembled closely to the 2-deox y-scyllo-inosose synthase in the Streptomyces fradiae.