Crystallographic Study of Molecular Mechanism of DNA Repair by Photolyase

光解酶是50 - 70 kDa单链蛋白，含有两种不同的等摩尔量的显色辅助因子。光再激活包括几个步骤：损伤识别和光解酶与DNA的结合、光子吸收、发色团间能量转移和电子从发色团到DNA的转移，从而导致紫外线诱导的嘧啶二聚体转化为单体。催化辅因子FAD对光依赖性修复过程至关重要。此外，存在第二个辅助因子，它作为光收集发色团。第二辅因子有两种已知结构，即存在于光解酶中的8-羟基-5-去氮黄素（8-HDF），如蓝藻、芽胞杆菌或大肠杆菌光解酶中发现的5,10-甲基四氢叶酸（MTHF）。利用同步辐射获得的x射线衍射数据，在1.8分辨率下测定了A.nidulans光解酶（53,000 Da）的晶体结构。由残基1 ~ 475、两个辅因子和192个水分子组成的精化模型的r因子为0.197。该结构由α / β和螺旋结构域组成，分别为8-HDF和FAD发色团提供结合位点。结果表明，8-HDF型光解酶的晶体结构与MTHF型大肠杆菌光解酶的骨架结构相似，但其捕光辅助因子的结合位点完全不同。这是密切相关蛋白的同源一级和三级结构在不同结合位点识别两种不同类型的辅因子的第一个例子。

Photolyases are 50 to 70 kDa single chain proteins containing two different chromophoric cofactors in equimolar amounts. Photoreactivation comprises several steps : damage recognition and binding of photolyase to DNA,photon absorption, interchromophoric energy transfer and electron transfer from chrompophore to DNA,resulting in the reversal of UV-induced pyrimidine dimers into monomers. The catalytic cofactor FAD is essential for the light-dependent repair process. In addition, a second cofactor is present which acts as a light-harvesting chromophore. Two structures are known for the second cofactor, either 8-hydroxy-5-deazaflavin (8-HDF) present in photolyase from e.g.the cyanobacterium, Anacystis nidulans or 5,10-methenyltetrahydro-folic acid (MTHF) found in E.coli photolyase. The crystal structure of A.nidulans photolyase (53,000 Da) was determined at 1.8 resolution from X-ray diffraction data obtained with synchrotron radiation. The refined model, comprising the residues 1 to 475, the two cofactors and 192 water molecules, has an R-factor of 0.197. The structure is composed of an alpha/beta and a helical domain, which provides binding sites for the 8-HDF and FAD chromophores, respectively. The present crystal structure of 8-HDF type photolyase from Anacystis nidulans showed the similarity of the backbone structure with MTHF type E.coli photolyase but completely different binding site of the light-harvesting cofactor. This is a first example that homologous primary and tertiary structures in closely related proteins recognize two different rtpes of cofactors at different binding-sites.