Structure and dynamics of novel bacterial photolyases

新型细菌光解酶的结构和动力学

• 批准号: 410476550
• 负责人: Professor Dr. Lars-Oliver Essen
• 金额: --
• 依托单位: National Natural Science Foundation of China
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/410476550?language=en
• 关键词: Structure; dynamics; novel; bacterial; photolyases

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.

光裂酶和隐花色素形成光驱动蛋白超家族（PCSf），存在于生命的所有领域。CPD和（6-4）光解酶修复UV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（-）光激发后。隐花色素赋予许多生物光响应，并依赖于已知的光解酶的黄素光还原过程。大多数光解酶和隐花色素通过保存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。相比之下，一些PCSf家族与这些典型的和研究充分的光解酶/隐花色素只有远亲关系。例如，细菌（6-4）光解酶（其至少在某些情况下也能够充当隐花色素）和II类CPD光解酶（CPDII）在DNA识别、电子转移途径和辅助光天线方面不同于真核生物（6-4）光解酶和其它CPD光解酶。 我们将探索由细菌（6-4）光解酶催化的DNA修复，结合我们的能力结构生物学，光生物学和时间分辨光谱学。在这里，我们将首先集中在PhrB从根癌农杆菌和其他完善的成员的家庭的细菌（6-4）的光解酶。为了解决他们的机制，在黄素光还原和6-4PP修复，我们采用瞬态吸收技术，具有非常宽的时间观测范围（100 fs到秒）和光谱覆盖范围。此外，我们将系统地应用定点诱变精细地图6-4PP修复过程中的电子和质子转移反应，以揭示否则模糊的反应途径。将通过X射线晶体学分析6-4PP-DNA与细菌（6-4）光解酶结合的模式以及突变对3D结构的影响。简明的机制模型与我们的光谱和结构数据一致，将描绘这种类型的（6-4）光解酶，并可能对我们的理解有深远的影响，以及真核生物（6-4）光解酶。 最后，PCSf的进化起源目前仍然没有解决。使用我们的结构和机制的知识的PCSf的成员，我们将描述一个新的PCSf家庭与预测的CPD光解酶活性。这个家族的成员缺乏N-末端天线域，否则严格保守的PCSf内。同时，我们将追求一个逆向工程策略，以产生和表征最小的单域光解酶从已知的PCSf成员。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的最早祖先，它们可能还不需要额外的蓝光捕获天线结构域，完全依赖于它们的FADH（−）发色团的光化学。