Kinetic isotope effect and reaction mechanism of 2-deoxy-scyllo-inosose synthase derived from butirosin-producing Bacillus circulans

Fumitaka Kudo, Noriaki Yamauchi, Rieko Suzuki, Katsumi Kakinuma

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Abstract

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, 3-18O]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 k(H)/k(D) = 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.

Original languageEnglish
Pages (from-to)424-428
Number of pages5
JournalJournal of Antibiotics
Volume50
Issue number5
Publication statusPublished - May 1 1997
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Drug Discovery

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