Jia Qian, Zeng Hui, Xiao Nan, Tang Jing, Gao Shangfang, Xie Wei
MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, Guangdong 510006, People's Republic of China.
Biochem J. 2025 Feb 12;482(4):BCJ20240728. doi: 10.1042/BCJ20240728.
The N6-methyladenine (6mA) modification is an essential epigenetic marker and plays a crucial role in processes, such as DNA repair, replication, and gene expression regulation. YerA from Bacillus subtilis is considered a novel class of enzymes that are capable of catalyzing the deamination of 6mA to produce hypoxanthine. Despite the significance of this type of enzymes in bacterial self-defense system and potential applications as a gene-editing tool, the substrate specificity, catalytic mechanism, and physiological function of YerA are currently unclear due to the lack of structural information. In the present study, we expressed the recombinant enzyme and conducted its reconstitution to yield the active form. Our deamination assays showed that N6-methyladenosine (N6-mAdo) served as a more favorable substrate than its base derivative 6mA. Here, we report the high-resolution structure of the C-terminal region of YerA, which exhibited a compact architecture composed of two antiparallel β-sheets with no obvious close structural homologs in Protein Data Bank. We also created docking models to investigate the ligand-binding pattern and found that more favorable contacts of N6-mAdo with the enzyme-binding pocket lead to its preference for N6-mAdo over 6mA. Finally, structural comparison of the N6-methyladenosine monophosphate deaminase allowed us to propose that a plausible role for this C-terminal region is to shield the active site from solvent and protect the intermediate during catalysis. Taken together, this study sheds light on the catalytic mechanism and evolutionary pathways of the promiscuous enzyme YerA, thereby contributing to our molecular understanding of epigenetic nucleoside metabolism.
N6-甲基腺嘌呤（6mA）修饰是一种重要的表观遗传标记，在DNA修复、复制和基因表达调控等过程中发挥着关键作用。枯草芽孢杆菌的YerA被认为是一类新型酶，能够催化6mA脱氨生成次黄嘌呤。尽管这类酶在细菌自我防御系统中具有重要意义，并且作为基因编辑工具具有潜在应用价值，但由于缺乏结构信息，YerA的底物特异性、催化机制和生理功能目前尚不清楚。在本研究中，我们表达了重组酶并进行了重组以产生活性形式。我们的脱氨分析表明，N6-甲基腺苷（N6-mAdo）比其碱基衍生物6mA是更有利的底物。在此，我们报道了YerA C端区域的高分辨率结构，其呈现出由两个反平行β-折叠组成的紧凑结构，在蛋白质数据库中没有明显的紧密结构同源物。我们还创建了对接模型来研究配体结合模式，发现N6-mAdo与酶结合口袋更有利的接触导致其对N6-mAdo的偏好超过6mA。最后，对N6-甲基腺苷单磷酸脱氨酶的结构比较使我们提出，这个C端区域的一个合理作用是在催化过程中保护活性位点免受溶剂影响并保护中间体。综上所述，本研究揭示了混杂酶YerA的催化机制和进化途径，从而有助于我们从分子层面理解表观遗传核苷代谢。
