Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase.
Studies on the mechanism of p-hydroxyphenylacetate 3-hydroxylase from Pseudomonas aeruginosa: a system composed of a small flavin reductase and a large flavin-dependent oxygenase.
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DOI：
10.1021/bi901454u
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发表时间：
2010-01-19
期刊：
影响因子：
2.9
通讯作者：
Ballou, David P.
中科院分区：
文献类型：
--
作者：
Chakraborty, Sumita;Ortiz-Maldonado, Mariliz;Entsch, Barrie;Ballou, David P.
有两种已知的微生物双组分黄素依赖的单加氧酶，它们催化对羟基苯乙酸酯(HPA)的氧化，它们的不同之处在于它们具有结构上不同的还原酶和加氧酶。本文详细分析了来自铜绿假单胞菌的酶的性质，并将其性质与发表的鲍曼不动杆菌酶的性质进行了比较，鲍曼不动杆菌是替代性的例子。铜绿假单胞菌的还原酶和加氧酶在大肠杆菌中得到了表达。将该还原酶纯化为含有弱结合FAD的稳定的C端His标记的黄色蛋白，将加氧酶纯化为稳定的无色N端His标记的蛋白。该还原酶催化FAD被NADH还原，并将FADH−产物释放到溶液中，但与鲍曼不动杆菌的还原酶不同，该催化作用不受HPA的影响。加氧酶与释放的FADH−结合，催化HPA氧化生成3，4-二羟基苯乙酸酯，然后FAD解离，被还原酶重新还原，这是两组分黄素依赖加氧酶的共同模式。在这个系统中，还原酶和加氧酶之间的相互作用似乎可以促进FADH−向加氧酶的转移，尽管它们不是必需的。我们发现，铜绿假单胞菌加氧酶系统在与FADH−的复合体中与O2反应形成准稳定的、异常高消光的黄素氢过氧化氢物种，该物种与HPA结合并反应形成产物。生成的黄素氢氧化物在仍与加氧酶结合的情况下分解成FAD和水，然后从蛋白质中释放出产物和FAD。与鲍曼不动杆菌的酶不同，在涉及还原酶和加氧酶的正常催化过程中，催化中的速率决定步骤是FAD从加氧酶中解离，这一过程与HPA的浓度无关。鲍曼不动杆菌和嗜热嗜热杆菌(类似于铜绿假单胞菌系统)的还原酶和加氧酶的结构构成了解释两组黄素依赖的双组分加氧酶差异的分子来源的基础。
There are two known types of microbial two-component flavin-dependent monooxygenases that catalyze oxygenation of p-hydroxyphenylacetate (HPA), and they are distinguished by having structurally distinct reductases and oxygenases. This paper presents a detailed analysis of the properties of the enzyme from Pseudomonas aeruginosa, an example of one group, and compares its properties to those published for the Acinetobacter baumannii enzyme, an example of the alternative group. The reductase and oxygenase from P. aeruginosa were expressed in Escherichia coli. The reductase was purified as a stable C-terminal His-tagged yellow protein containing weakly bound FAD, and the oxygenase was purified as a stable colorless N-terminal His-tagged protein. The reductase catalyzes the reduction of FAD by NADH and releases the FADH− product into solution, but unlike the reductase from A. baumannii, this catalysis is not influenced by HPA. The oxygenase binds the released FADH− and catalyzes the oxygenation of HPA to form 3,4-dihydroxyphenylacetate, after which the FAD dissociates to be re-reduced by the reductase, a common overall pattern for two-component flavin-dependent oxygenases. With this system, it appears that interactions between the reductase and the oxygenase can facillitate the transfer of FADH− to the oxygenase, although they are not required. We show that the P. aeruginosa oxygenase system in complex with FADH− reacts with O2 to form a quasi-stable, unusually high-extinction flavin hydroperoxide species that binds HPA and reacts to form the product. The resultant flavin hydroxide decomposes to FAD and water while still bound to the oxygenase, and then releases product and FAD from the protein. Unlike the enzyme from A. baumannii, during normal catalysis involving both the reductase and oxygenase, the rate-determining step in catalysis is the dissociation of FAD from the oxygenase in a process that is independent of the concentration of HPA. Structures for the reductases and oxygenases from A. baumannii and from Thermus thermophilus (similar to the P. aeruginosa system) form a basis for interpreting the molecular origins of the differences between the two groups of flavin-dependent two-component oxygenases.
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影响因子：
4.8
作者：
Campbell, Zachary T.;Baldwin, Thomas O.
通讯作者：
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DOI：
10.1073/pnas.0800859105
发表时间：
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