Structure and dynamics of novel bacterial photolyases

光解酶和隐花色素形成了一个光驱动蛋白质超家族(PCSf)，存在于生命的各个领域。CPD和(6-4)光解酶修复紫外线诱导的DNA损伤，环丁烷嘧啶二聚体(CPD)和(6-4)光产物(6-4PP)在其完全还原的黄素腺嘌呤二核苷酸辅因子FADH(-)的光激发下修复。隐色素提供许多生物光反应，并依赖于已知的黄素光解酶的光还原过程。大多数光解酶和隐色素酶通过保留其光活性区域的主要结构和功能特征而具有密切的进化关系。相反，一些PCSf家族只与这些典型的和研究得很好的光解酶/隐花色素有远亲关系。例如，细菌(6-4)光解酶和第二类CPD光解酶(CPDII)在DNA识别、电子转移途径和辅助光天线方面与真核(6-4)光解酶和其他CPD光解酶不同。细菌(6-4)光解酶至少在某些情况下也能作为隐色素发挥作用。我们将结合我们的结构生物学、光生物学和时间分辨光谱学的能力，探索细菌(6-4)光解酶催化的DNA修复。在这里，我们首先将重点放在根癌农杆菌和细菌(6-4)光解酶家族中其他成熟成员的PhrB上。为了从黄素光还原和6-4PP修复的角度研究它们的机制，我们使用了具有异常广泛的时间观察(100飞秒到秒)和光谱覆盖范围的瞬时吸收技术。此外，我们将系统地应用定点突变来精细映射6-4PP修复过程中的电子和质子转移反应，以揭示其他被掩盖的反应途径。6-4PP-DNA与细菌(6-4)光解酶的结合方式以及突变对3D结构的影响将通过X射线结晶学进行分析。与我们的光谱和结构数据相一致的简明机制模型将为这种类型的(6-4)光解酶描绘出来，并可能对我们对真核(6-4)光解酶的理解产生深远的影响。最后，PCSf的进化起源目前仍未解决。利用我们对PCSf成员的结构和机制知识，我们将描述一个新的具有预测的CPD光解酶活性的PCSf家族。这个家族的成员缺乏N端天线结构域，否则在PCSf中严格保守。同时，我们将采用反向工程策略，从已知的PCSf成员中产生和表征最小的单域光解酶。这种方法将解决PCSf的蛋白质进化问题，并可能引导我们找到PCSf的最早的祖先，这可能还不需要额外的蓝光捕获天线结构域，并且完全依赖于它们的FADH(−)发色团的光化学。

Photolyases and cryptochromes form a superfamily (PCSf) of light-driven proteins occurring in all domains of Life. CPD and (6-4) photolyases repair UV induced DNA lesions, cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP), after photoexcitation of their fully reduced flavin adenine dinucleotide cofactor, FADH(-). Cryptochromes confer many biological light responses and depend for that on the flavin photoreduction processes known from photolyases. Most photolyases and cryptochromes have intimate evolutionary relationships by conserving major structural and functional features of their photoactive regions. In contrast, some PCSf families are only distantly related to these canonical and well-studied photolyases/cryptochromes. For example, bacterial (6-4) photolyases, which are at least in some cases capable to act as cryptochromes as well, and class II CPD photolyases (CPDII) differ from eukaryotic (6-4) photolyases and other CPD photolyases in terms of DNA recognition, electron transfer pathways and auxillary photoantennas. We will explore DNA repair catalyzed by bacterial (6-4) photolyases by bundling our competences structural biology, photobiology and time-resolved spectroscopy. Here, we will first focus on PhrB from Agrobacterium tumefaciens and other well-established members of the family of bacterial (6-4) photolyases. To address their mechanisms in terms of flavin photoreduction and 6-4PP repair we employ transient absorption techniques with exceptionally wide ranges of temporal observation (100 fs to seconds) and spectral coverage. Additionally, we will systematically apply site-directed mutagenesis to fine-map electron and proton transfer reactions during 6-4PP repair to reveal otherwise obscured reaction pathways. The mode of 6-4PP-DNA binding to bacterial (6-4) photolyases and the effect of mutations on the 3D structure will be analyzed by X-ray crystallography. Concise mechanistic models consistent with our spectroscopic and structural data will be delineated for this type of (6-4) photolyases and may have profound influence on our understanding of eukaryotic (6-4) photolyases as well. Finally, the evolutionary origin of the PCSf is currently still unresolved. Using our structural and mechanistic knowledge on members of the PCSf we will characterize a novel PCSf family with predicted CPD photolyase activity. Members of this family lack the N-terminal antenna domain otherwise strictly conserved within the PCSf. In parallel, we will pursue a reverse-engineering strategy to generate and characterize minimal single-domain photolyases from known PCSf members. This approach will address the protein evolution of the PCSf and may lead us to the earliest ancestors of the PCSf, which may not have yet required an additional blue-light harvesting antenna domain and depended entirely on the photochemistry of their FADH(−) chromophore.