Hasan N, Nester E W
J Biol Chem. 1978 Jul 25;253(14):4987-92.
NADPH-dependent flavin reductase (required for the activation of chorismate synthase) was purified to homogeneity from cell-free extracts of Bacillus subtilis. The enzyme has a molecular weight of 13,000 as determined by sodium dodecyl sulfate-gel electrophoresis, is specific for NADPH, and requires a divalent metal ion and either FMN or FAD for maximal rates of NADPH oxidation. The enzyme is able to reduce 2,6-dichlorophenolindophenol (DCIP) in the presence of NADPH and a divalent metal ion. Both catalytic activities were completely inhibited by EDTA. The Km for FMN is 1.25 X 10(-5) M and for NADPH 7.8 X 10(-5) M with oxygen as the final electron acceptor, and 3.85 X 10(-4) M with DCIP as the final electron acceptor. The enzyme was also isolated in association with chorismate synthase and dehydroquinate synthase. The enzyme associated with the complex has the same catalytic properties as the dissociated enzyme except that it requires both a divalent metal ion and FMN for DCIP reduction. Maximal enzyme activity was observed when the enzyme was preincubated with FMN and the divalent metal ion. The enzyme complex is easily dissociable and the dissociation of the enzyme complex resulted in the failure of NADPH-dependent flavin reductase to adsorb to phosphocellulose.
从枯草芽孢杆菌的无细胞提取物中纯化出了NADPH依赖性黄素还原酶（分支酸合酶激活所必需的），使其达到了均一性。通过十二烷基硫酸钠-凝胶电泳测定，该酶的分子量为13,000，对NADPH具有特异性，并且需要二价金属离子以及FMN或FAD才能实现NADPH氧化的最大速率。在存在NADPH和二价金属离子的情况下，该酶能够还原2,6-二氯酚靛酚（DCIP）。两种催化活性均被EDTA完全抑制。以氧气作为最终电子受体时，FMN的Km为1.25×10⁻⁵ M，NADPH的Km为7.8×10⁻⁵ M；以DCIP作为最终电子受体时，NADPH的Km为3.85×10⁻⁴ M。该酶还与分支酸合酶和脱氢奎尼酸合酶一起被分离出来。与复合物结合的酶具有与解离后的酶相同的催化特性，只是它在还原DCIP时需要二价金属离子和FMN两者。当该酶与FMN和二价金属离子预孵育时，观察到了最大酶活性。酶复合物很容易解离，酶复合物的解离导致NADPH依赖性黄素还原酶无法吸附到磷酸纤维素上。
