Replacement of non-heme Fe(II) with Cu(II) in the α-ketoglutarate dependent DNA repair enzyme AlkB:: Spectroscopic characterization of the active site
Replacement of non-heme Fe(II) with Cu(II) in the α-ketoglutarate dependent DNA repair enzyme AlkB:: Spectroscopic characterization of the active site
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DOI：
10.1016/j.jinorgbio.2007.03.018
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发表时间：
2007-07-01
影响因子：
3.9
通讯作者：
Matthews, Steve J.
中科院分区：
文献类型：
--
作者：
Bleijlevens, Boris;Shivarattan, Tara;Matthews, Steve J.
细菌DNA修复酶AlkB是一种α-酮戊二酸（α KG）依赖性非血红素Fe（II）双加氧酶。在这里，我们描述，第一次，制备的Cu（II）-重构形式的AlkB在各种配合物。光谱表征表明正确的AlkB折叠后，纳入Cu（II）的活性位点。通过EPR光谱将Cu位点归类为2型位点。活性中心金属的可及性进行了研究，使用咪唑作为探针。虽然咪唑的加入没有改变AlkB-Cu-α KG络合物的EPR谱，但AlkB-Cu-琥珀酸盐络合物的谱明显改变，表明咪唑在Cu位点的结合。底物（甲基化DNA）与AlkB-Cu-alpha KG复合物的结合不会引起EPR谱的变化，这表明底物不会与金属中心紧邻。这项工作提供了先进的EPR方法，旨在研究AlkB复合物在溶液中的相互作用和动力学的基础。(c)2007年爱思唯尔公司All rights reserved.
The bacterial DNA repair enzyme AlkB is an alpha-ketoglutarate (alpha KG) dependent non-heme Fe(II) containing dioxygenase. Here we describe, for the first time, the preparation of a Cu(II)-reconstituted form of AlkB in various complexes. Spectroscopic characterization showed correct AlkB folding upon incorporation of Cu(II) in the active site. The Cu site was classified as a type 2 site by EPR spectroscopy. The accessibility of the active site metal was studied using imidazole as a probe. Although addition of imidazole did not change the EPR spectrum of the AlkB-Cu-alpha KG complex, the spectrum of the AlkB-Cu-succinate complex clearly changed, indicating binding of imidazole at the Cu site. Binding of substrate (methylated DNA) to the AlkB-Cu-alpha KG complex did not induce changes in the EPR spectrum, demonstrating that the substrate does not bind in the immediate vicinity of the metal centre. This work provides a basis for advanced EPR approaches aimed at studying the interactions and dynamics of AlkB complexes in solution. (c) 2007 Elsevier Inc. All rights reserved.