Abstract

Photolyases are flavoproteins that repair ultraviolet-induced DNA lesions (cyclobutane pyrimidine dimer or CPD, and pyrimidine (6-4) pyrimidone photoproducts or (6-4)-PPs), using blue light as an energy source. These enzymes are substrate specific, meaning that a specific photolyase repairs either a CPD or a (6-4)-PP. In this work, we produced a class II CPD-photolyase (called as PhrSph98) from the Antarctic bacterium Sphingomonas sp. UV9 by recombinant DNA technology and we purified the enzyme using immobilized metal affinity chromatography. By using an immunochemistry assay, with monoclonal antibodies against CPD and (6-4)-PP, we found that PhrSph98 repairs both DNA lesions. The result was confirmed by immunocytochemistry using immortalized non-tumorigenic human keratinocytes. Results from structure prediction, pocket computation, and molecular docking analyses showed that PhrSph98 has the two expected protein domains (light-harvesting antenna and a catalytic domain), a larger catalytic site as compared with photolyases produced by mesophilic organisms, and that both substrates fit the catalytic domain. The results obtained from predicted homology modeling suggest that the electron transfer pathway may occur following this pathway: Y389-W369-W390-F376-W381/FAD. The evolutionary reconstruction of PhrSph98 suggests that this is a missing link that reflects the transition of (6-4)-PP repair into the CPD repair ability for the class II CPD-photolyases. To the best of our knowledge, this is the first report of a naturally occurring bifunctional, CPD and (6-4)-PP, repairing enzyme.

Key points

• We report the first described bifunctional CPD/(6-4)-photoproducts repairing enzyme. The bifunctional enzyme reaches the nuclei of keratinocyte and repairs the UV-induced DNA damage. The enzyme should be a missing link from an evolutionary point of view. The enzyme may have potential uses in the pharmaceutical and cosmetic industries.

中文翻译：

---

天然存在的双功能CPD /（6-4）-光解酶，来自南极细菌鞘氨醇单胞菌（Sphingomonas sp。）。紫外线9。

摘要

光解酶是一种黄素蛋白，可以利用蓝光作为能量来修复紫外线诱发的DNA损伤（环丁烷嘧啶二聚体或CPD，以及嘧啶（6-4）嘧啶光产物或（6-4）-PPs）。这些酶是底物特异性的，这意味着特定的光解酶可以修复CPD或（6-4）-PP。在这项工作中，我们从南极细菌鞘氨醇单胞菌（Sphingomonas）生产了II类CPD-光解酶（称为PhrSph98）sp。通过重组DNA技术获得UV9，我们使用固定的金属亲和色谱法纯化了该酶。通过使用免疫化学分析，针对CPD和（6-4）-PP的单克隆抗体，我们发现PhrSph98修复了两个DNA损伤。使用永生化的非致瘤性人角质形成细胞通过免疫细胞化学证实了该结果。结构预测，口袋计算和分子对接分析的结果表明，PhrSph98具有两个预期的蛋白质结构域（光捕获天线和一个催化结构域），与中温生物产生的光裂解酶相比，催化位点更大，并且两种底物都适合催化域。从预测的同源性建模获得的结果表明，电子转移途径可能遵循该途径：Y389-W369-W390-F376-W381 / FAD。PhrSph98的进化重建表明，这是一个缺失的环节，反映了（6-4）-PP修复向II类CPD-光解酶的CPD修复能力的转变。据我们所知，这是天然存在的双功能CPD和（6-4）-PP修复酶的首次报道。

关键点

•我们报告了第一个描述的双功能CPD /（6-4）-光产物修复酶。双功能酶到达角质形成细胞的核并修复紫外线诱导的DNA损伤。从进化的角度来看，该酶应该是一个缺失的环节。该酶在制药和化妆品工业中可能具有潜在用途。