Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases, College of Life Sciences, Anhui Normal University, Wuhu, Anhui, China.
Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
J Biol Chem. 2022 Aug;298(8):102188. doi: 10.1016/j.jbc.2022.102188. Epub 2022 Jun 24.
The UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 photoproducts), can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. The fully reduced flavin (hydroquinone, HQ) cofactor is required for the catalysis of both types of these photolyases. On the other hand, flavin cofactor in the semireduced state, semiquinone, can be utilized by photolyase homologs, the cryptochromes. However, the evolutionary process of the transition of the functional states of flavin cofactors in photolyases and cryptochromes remains mysterious. In this work, we investigated three representative photolyases (Escherichia coli CPD photolyase, Microcystis aeruginosa DASH, and Phaeodactylum tricornutum 6-4 photolyase). We show that the residue at a single site adjacent to the flavin cofactor (corresponding to Ala377 in E. coli CPD photolyase, hereafter referred to as site 377) can fine-tune the stability of the HQ cofactor. We found that, in the presence of a polar residue (such as Ser or Asn) at site 377, HQ was stabilized against oxidation. Furthermore, this polar residue enhanced the photorepair activity of these photolyases both in vitro and in vivo. In contrast, substitution of hydrophobic residues, such as Ile, at site 377 in these photolyases adversely affected the stability of HQ. We speculate that these differential residue preferences at site 377 in photolyase proteins might reflect an important evolutionary event that altered the stability of HQ on the timeline from expression of photolyases to that of cryptochromes.
紫外线诱导的 DNA 损伤，环丁烷嘧啶二聚体（CPD）和嘧啶（6-4）嘧啶酮光产物（6-4 光产物），可以分别被 CPD 光解酶和 6-4 光解酶直接光修复。完全还原的黄素（氢醌，HQ）辅因子是这两种光解酶催化所必需的。另一方面，黄素辅因子在半还原状态，半醌，可以被光解酶同源物，隐色素利用。然而，黄素辅因子在光解酶和隐色素中的功能状态的转变的进化过程仍然是神秘的。在这项工作中，我们研究了三种代表性的光解酶（大肠杆菌 CPD 光解酶、微囊藻 DASH 和三角褐指藻 6-4 光解酶）。我们表明，黄素辅因子附近的单个位置的残基（对应于大肠杆菌 CPD 光解酶中的 Ala377，以下称为位置 377）可以微调 HQ 辅因子的稳定性。我们发现，在位置 377 存在极性残基（如 Ser 或 Asn）时，HQ 对氧化稳定。此外，这种极性残基增强了这些光解酶在体外和体内的光修复活性。相比之下，在这些光解酶中的位置 377 取代疏水性残基，如 Ile，会不利地影响 HQ 的稳定性。我们推测，光解酶蛋白中位置 377 处的这些差异残基偏好可能反映了一个重要的进化事件，该事件改变了 HQ 在从光解酶表达到隐色素表达的时间线上的稳定性。
