Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF
Structure/function correlations among coupled binuclear copper proteins through spectroscopic and reactivity studies of NspF
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DOI：
10.1073/pnas.1208718109
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发表时间：
2012-07-03
影响因子：
11.1
通讯作者：
Solomon, Edward I.
中科院分区：
文献类型：
--
作者：
Ginsbach, Jake W.;Kieber-Emmons, Matthew T.;Solomon, Edward I.
4-羟基-3-亚硝基苯甲酰胺在村山链霉菌中生物合成的最终步骤由NspF进行，NspF是一种单加氧酶，可将邻氨基苯酚转化为相应的亚硝基产物（羟基苯胺酶活性）。以前的生化表征的静息形式的NspF表明，这种酶属于耦合双核铜酶（CBC）的家庭。该酶家族的另一个成员酪氨酸酶能够单-还原单酚（单酚酶活性），但不能还原邻氨基酚。为了深入了解NspF的独特反应性，我们已经生成并表征了其活性位点的氧形式。与氧化酪氨酸酶相同的光谱特征的观察表明，oxy-NspF含有Cu 2 O2核心，其中过氧化物以μ-η（2）：η（2）模式配位，证实NspF是CBC酶。发现这种氧合形式与单酚反应，表明与酪氨酸酶一样，NspF也具有单酚酶活性。的monophenolase和羟基苯胺酶活性的两个亲电机制的比较表明，它们各自的过渡态之间的一个大的几何变化。在每个过渡态的蛋白质口袋和基板之间的特定相互作用的潜力进行了讨论的上下文中的差分反应性的这个家庭的酶与等效的μ-η（2）：η（2）过氧桥耦合双核铜活性位点。
The terminal step of 4-hydroxy-3-nitrosobenzamide biosynthesis in Streptomyces murayamaensis is performed by NspF, a mono-oxygenase that converts o-aminophenols to the corresponding nitroso product (hydroxyanilinase activity). Previous biochemical characterization of the resting form of NspF suggested that this enzyme belonged to the coupled binuclear copper enzyme (CBC) family. Another member of this enzyme family, tyrosinase, is able to mono-oxygenate monophenols (monophenolase activity) but not o-aminophenols. To gain insight into the unique reactivity of NspF, we have generated and characterized the oxy form of its active site. The observation of spectral features identical to those of oxytyrosinase indicates that oxy-NspF contains a Cu2O2 core where peroxide is coordinated in a mu-eta(2):eta(2) mode, confirming that NspF is a CBC enzyme. This oxy form is found to react with monophenols, indicating that, like tyrosinase, NspF also possesses monophenolase activity. A comparison of the two electrophilic mechanisms for the monophenolase and hydroxyanilinase activity indicates a large geometric change between their respective transition states. The potential for specific interactions between the protein pocket and the substrate in each transition state is discussed within the context of the differential reactivity of this family of enzymes with equivalent mu-eta(2):eta(2) peroxy bridged coupled binuclear copper active sites.