Yamada Daichi, Yamamoto Junpei, Zhang Yu, Iwata Tatsuya, Hitomi Kenichi, Getzoff Elizabeth D, Iwai Shigenori, Kandori Hideki
Department of Frontier Materials, Nagoya Institute of Technology , Showa-ku, Nagoya 466-8555, Japan.
Graduate School of Engineering Science, Osaka University , Toyonaka, Osaka 560-8531, Japan.
Biochemistry. 2016 Feb 2;55(4):715-23. doi: 10.1021/acs.biochem.5b01111. Epub 2016 Jan 19.
Photolyases (PHRs) repair the UV-induced photoproducts, cyclobutane pyrimidine dimer (CPD) or pyrimidine-pyrimidone (6-4) photoproduct [(6-4) PP], restoring normal bases to maintain genetic integrity. CPD and (6-4) PP are repaired by substrate-specific PHRs, CPD PHR and (6-4) PHR, respectively. Flavin adenine dinucleotide (FAD) is the chromophore of both PHRs, and the resting oxidized form (FAD(ox)), at least under in vitro purified conditions, is first photoconverted to the neutral semiquinoid radical (FADH(•)) form, followed by photoconversion into the enzymatically active fully reduced (FADH(-)) form. Previously, we reported light-induced difference Fourier transform infrared (FTIR) spectra corresponding to the photoactivation process of Xenopus (6-4) PHR. Spectral differences between the absence and presence of (6-4) PP were observed in the photoactivation process. To identify the FTIR signals where these differences appeared, we compared the FTIR spectra of photoactivation (i) in the presence and absence of (6-4) PP, (ii) of (13)C labeling, (15)N labeling, and [(14)N]His/(15)N labeling, and (iii) of H354A and H358A mutants. We successfully assigned the vibrational bands for (6-4) PP, the α-helix and neutral His residue(s). In particular, we assigned three bands to the C ═ O groups of (6-4) PP in the three different redox states of FAD. Furthermore, the changed hydrogen bonding environments of C ═ O groups of (6-4) PP suggested restructuring of the binding pocket of the DNA lesion in the process of photoactivation.
光解酶（PHRs）可修复紫外线诱导产生的光产物，即环丁烷嘧啶二聚体（CPD）或嘧啶 - 嘧啶酮（6 - 4）光产物[（6 - 4）PP]，通过恢复正常碱基来维持基因完整性。CPD和（6 - 4）PP分别由底物特异性光解酶CPD光解酶和（6 - 4）光解酶修复。黄素腺嘌呤二核苷酸（FAD）是这两种光解酶的发色团，至少在体外纯化条件下，静止的氧化形式（FAD(ox)）首先被光转化为中性半醌自由基（FADH(•)）形式，随后再光转化为酶活性的完全还原形式（FADH(-)）。此前，我们报道了非洲爪蟾（6 - 4）光解酶光激活过程对应的光诱导差示傅里叶变换红外（FTIR）光谱。在光激活过程中观察到有无（6 - 4）PP时光谱的差异。为了确定出现这些差异的FTIR信号，我们比较了以下几种情况下光激活的FTIR光谱：（i）有无（6 - 4）PP；（ii）(13)C标记、(15)N标记以及[(14)N]His/(15)N标记；（iii）H354A和H358A突变体。我们成功归属了（6 - 4）PP、α - 螺旋和中性组氨酸残基的振动谱带。特别是，我们在FAD的三种不同氧化还原状态下，将三条谱带归属为（6 - 4）PP的C ═ O基团。此外，（6 - 4）PP的C ═ O基团氢键环境的变化表明在光激活过程中DNA损伤结合口袋发生了结构重组。
