Identification and characterization of a second chromophore of DNA photolyase from Thermus thermophilus HB27
Identification and characterization of a second chromophore of DNA photolyase from Thermus thermophilus HB27
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DOI：
10.1074/jbc.m507972200
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发表时间：
2005-10-28
影响因子：
4.8
通讯作者：
Shimada, I
中科院分区：
文献类型：
--
作者：
Ueda, T;Kato, A;Shimada, I
环丁烷嘧啶二聚体（CPD）光解酶利用光来修复CPD。为了有效的光吸收，CPD光解酶使用第二生色团。我们纯化了嗜热栖热菌CPD光裂合酶及其第二发色团。紫外-可见吸收光谱，反相高效液相色谱法，和NMR分析的发色团显示，第二发色团的酶是黄素单甘肽（FMN）。为了阐明FMN在CPD修复反应中的作用，成功地获得了不含FMN的酶（Enz-FMN（-））和具有化学计量量的FMN的酶（Enz-FMN（+））。酶FMN（+）的CPD修复活性高于酶FMN（-），且酶FMN（+）和酶FMN（-）的CPD修复活性比值与光的波长有关。这些结果表明，FMN增加了酶的光吸收效率。对Enz-FMN（+）和Enz-FMN（-）的NMR分析表明，FMN与Anacystis nidulans CPD光解酶中7，8-二甲基-8-羟基-5-脱氮核黄素的结合方式相似，因此FMN与CPD之间不可能发生直接的电子转移。基于这些结果，我们得出结论，FMN作为一种高效的光收集器，收集光并将能量转移到FAD。
Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN ( Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.