A uracil DNA glycosylase (UDG) from Mycobacterium smegmatis (MsmUdgX) shares sequence similarity with family 4 UDGs and forms exceedingly stable complexes with single-stranded uracil-containing DNAs (ssDNA-Us) that are resistant to denaturants. However, MsmUdgX has been reported to be inactive in excising uracil from ssDNA-Us and the underlying structural basis is unclear. Here, we report high-resolution crystal structures of MsmUdgX in the free, uracil- and DNA-bound forms, respectively. The structural information, supported by mutational and biochemical analyses, indicates that the conserved residue His109 located on a characteristic loop forms an irreversible covalent linkage with the deoxyribose at the apyrimidinic site of ssDNA-U, thus rendering the enzyme unable to regenerate. By proposing the catalytic pathway and molecular mechanism for MsmUdgX, our studies provide an insight into family 4 UDGs and UDGs in general.

来自耻垢分枝杆菌（Msm UdgX）的尿嘧啶DNA糖基化酶（UDG）与家族4 UDG具有序列相似性，并与单链含尿嘧啶的DNA（ssDNA-Us）形成极其稳定的复合物，该DNA对变性剂具有抗性。但是，据报道，Msm UdgX在从ssDNA-Us切下尿嘧啶方面没有活性，其潜在的结构基础尚不清楚。在这里，我们报告Msm的高分辨率晶体结构UdgX分别呈游离形式，尿嘧啶结合形式和DNA结合形式。结构信息得到突变和生化分析的支持，表明位于特征环上的保守残基His109在ssDNA-U的嘧啶二烯位与脱氧核糖形成不可逆的共价键，从而使该酶无法再生。通过提出Msm UdgX的催化途径和分子机制，我们的研究提供了对4类UDG和一般UDG的深入了解。