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 carbocycle formation step from D-glucose-6-phosphate in the biosynthesis of the 2-deoxystreptamine aglycon of clinically important aminocyclitol antibiotics, was investigated with a partially purified enzyme from butirosin-producing Bacillus circulans SANK 72073, Nonlabeled and double-labeled D-[4-2H, 3-15O]glucose-6-phosphate were used for cross-over experiment, and the oxime-TMS ether derivative of the 2-deoxy-scyllo-inosose product was analyzed by GC-MS. The deuterium label at C-4 of the substrate appeared to be retained at C-6 of the inosose product without scrambling of the double-labeled isotopes. Since the transient reduction of NAD+ cofactor was proved to be essential in the 2-deoxy-scyllo-inosose reaction, the hydride abstraction and returning appeared to take place within the same glucose molecule. The observed kinetic isotope effect was estimated to be kH/kD = 2.4. These results strongly suggest that this carbocycle formation is catalyzed by a single 2-deoxy-scyllo-inosose synthase enzyme with catalytic requirement of NAD+, the mechanism of which appears to be resembled closely to the 2-deoxy-scyllo-inosose synthase in the Streptomyces fradiae.
Kudo
F
F
Department of Chemistry, Tokyo Institute of Technology, Japan.
Yamauchi
N
N
Suzuki
R
R
Kakinuma
K
K
D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D001407:Bacillus / Q000378:metabolism; D002076:Butirosin Sulfate / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006595:Hexosamines / Q000096:biosynthesis; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism / Q000493:pharmacokinetics*
D000900:Anti-Bacterial Agents; D006595:Hexosamines; D002076:Butirosin Sulfate; D008190:Lyases; C085631:2-deoxy-scyllo-inosose synthase; C007265:2-deoxystreptamine