5 2-デオキシストレプタミン生合成の初発反応を担う2-デオキシ-scyllo-イノソース合成酵素の精製と反応機構(口頭発表の部)

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2-Deoxystreptamine 1 is a common central aglycon in the major group of clinically important aminocyclitol antibiotics. The initial step of 2-deoxystreptamine biosynthesis is the intramolecular C-C bond formation between C-1 and C-6 of D-glucose-6-phosphate 3 to form 2-deoxy-scyllo-inosose 2 by 2-deoxy-scyllo-inosose synthase. An aldol-type cyclization involving transient oxidation of C-4 with NAD^+ and subsequent elimination of inorganic phosphate has been proposed. In this study, enzyme purification was carried out from centrifuged supernatant of the disrupted Bacillus circulans cells by ammonium sulfate fractionation, followed by DEAE-, Dye-ligand-, Q-Sepharose FF-, Mono Q- (x2) and gel filtration chromatography to afford two major spieces. The molecular weight of these enzymes were estimated to be about 45,000. Cross-over experiments with doubly labeled D-[4-^2H, 3-^<18>O] glucose-6-phosphate 1 1 were performed with the partially purified enzymes. The deuterium label at C-4 of the substrate was retained at C-6 of the product without scrambling the doubly-labeled isotope, and kinetic isotope effect (k_H/k_D ≒2.4-2.7) were observed in the enzyme reaction. These results strongly suggest that "2-deoxy-scyllo-inosose synthase" is a single enzyme, which catalyzes multistep reaction involving oxidation-reduction with NAD^+ cofactor and aldol-type carbocycle formation. Further, reaction with deoxy-G-6-P was investigated to clarify whether or not the other hydroxyl groups of G-6-P are participated in the reaction. Both 2-deoxy- and 3-deoxy-G-6-P were converted to some extent into their corresponding cyclitols. This indicates that the C-2 and C-3 hydroxyl groups are involved in the substrates recognition, but are not necessary for triggering the reaction.
天然有機化合物討論会の論文
1996-09-02

5 2-デオキシストレプタミン生合成の初発反応を担う2-デオキシ-scyllo-イノソース合成酵素の精製と反応機構(口頭発表の部)

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