NAPHTHALENE DIOXYGENASE - PURIFICATION AND PROPERTIES OF A TERMINAL OXYGENASE COMPONENT
NAPHTHALENE DIOXYGENASE - PURIFICATION AND PROPERTIES OF A TERMINAL OXYGENASE COMPONENT
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DOI：
10.1128/jb.155.2.505-511.1983
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发表时间：
1983-01-01
影响因子：
3.2
通讯作者：
GIBSON, DT
中科院分区：
文献类型：
--
作者：
ENSLEY, BD;GIBSON, DT
假单胞菌NCIB 9816的萘双加氧酶是一种多组分酶体系，可将萘氧化为顺式-(1R, 2S)-二羟基-1,2-二氢萘。末端加氧酶组分B通过离子交换和疏水相互作用色谱法纯化至均匀性。纯化后的酶仅在NADH， O2和部分纯化的组分A和c的存在下氧化萘，通过凝胶过滤得到的分子量估计为158,000。在十二烷基硫酸钠存在下，聚丙烯酰胺凝胶电泳显示存在2个亚基，分子量为。apprx。5万5千和2万，表明是。alpha。2。beta。2 .四元结构。氧化酶的吸收光谱在566（肩）、462和344 nm处达到最大值，在萘双加氧酶体系的其他两种组分存在的情况下，用化学计量量的NADH厌氧还原酶时，吸收光谱在520和380 nm处达到最大值。组分B结合萘。在添加组分A和C、NADH和O2后，酶结合的萘被氧化为产物。这些结果，再加上纯化酶的6.0 g铁原子和4.0 g酸不稳定S/mol原子的存在，表明萘双加氧酶系统的组分B是一种Fe-S蛋白，在萘氧化的最终步骤中起作用。
Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-diohydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a 3-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, O2 and partially purified preparations of components A and C. An estimated MW of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of 2 subunits with MW of .apprx. 55,000 and 20,000, indicative of an .alpha.2.beta.2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462 and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other 2 components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH and O2. These results, together with the detection of the presence of 6.0 g-atoms of Fe and 4.0 g-atoms of acid-labile S/mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an Fe-S protein which functions in the terminal step of naphthalene oxidation.