Crystallographic Study of Molecular Mechanism of DNA Repair by Photolyase

光解酶是50至70 kDa的单链蛋白，含有等摩尔量的两种不同的发色辅因子。光复活包括几个步骤：损伤识别和光解酶与DNA的结合，光子吸收，发色团间的能量转移和电子转移从发色团到DNA，导致UV诱导的嘧啶二聚体逆转为单体。催化辅因子FAD对于光依赖性修复过程是必不可少的。此外，存在第二辅因子，其充当捕光发色团。已知第二辅因子有两种结构，一种是存在于来自例如蓝细菌Anacystis nidulans的光裂合酶中的8-羟基-5-脱氮黄素（8-HDF），另一种是存在于大肠杆菌光裂合酶中的5，10-亚甲基四氢叶酸（MTHF）。从同步辐射获得的X射线衍射数据中以1.8的分辨率确定构巢曲霉光解酶（53，000 Da）的晶体结构。改进的模型，包括残基1至475，两个辅因子和192个水分子，具有0.197的R因子。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。目前的晶体结构显示8-HDF型光裂合酶的主链结构与MTHF型大肠杆菌光裂合酶相似，但捕光辅助因子的结合位点完全不同。这是第一个例子，同源的一级和三级结构在密切相关的蛋白质识别两个不同类型的辅因子在不同的结合位点。

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.