UdgX from Mycobacterium smegmatis (MsmUdgX) is a prototypical enzyme representing a new class of uracil-DNA glycosylases (UDG) closely related to the family 4 enzymes. It possesses a unique R-loop rich in positive residues and forms a covalent bond with single-stranded uracil-containing DNAs (ssDNA-Us) that is resistant to denaturants after the removal of the target uracil. We previously identified the H109E mutant of MsmUdgX that forms a weak covalent complex with ssDNA-U and yet possesses moderate uracil excision activity, but the mechanism of its action is not fully understood. To further study the catalytic process of MsmUdgX, we solved the high-resolution crystal structures of H109E in the free and DNA-bound forms, respectively. We found that the key residue Glu109 adopts a similar conformation to that of WT to form the covalent bond, suggesting that it still employs the same “excision-inhibition” mechanism to that of the WT enzyme. The enzyme remains nearly intact before and after the crosslinking reaction, but the first half of the R-loop exhibits large structural differences while the rest of the loop barely moves, owing to the salt-bridge interaction formed via Arg107. Additionally, Arg107, along with Gln53 was found to play important roles in the biochemical properties of MsmUdgX. Our studies provide new insights into the MsmUdgX catalysis and improve our understanding on this unique enzyme.

UdgX从耻垢分枝杆菌（MSM UdgX）是代表一类新的尿嘧啶DNA糖基化酶（UDG）密切相关的家族4种酶的典型酶。它拥有一个独特的富含正残基的 R 环，并与单链含尿嘧啶的 DNA (ssDNA-Us) 形成共价键，在去除目标尿嘧啶后对变性剂具有抗性。我们之前确定了Msm UdgX的 H109E 突变体，它与 ssDNA-U 形成弱共价复合物，但具有中等的尿嘧啶切除活性，但其作用机制尚不完全清楚。进一步研究MSM的催化过程UdgX，我们分别解析了游离和 DNA 结合形式的 H109E 的高分辨率晶体结构。我们发现关键残基Glu109采用与WT相似的构象形成共价键，表明它仍然采用与WT酶相同的“切除-抑制”机制。该酶在交联反应前后几乎保持完整，但 R 环的前半部分表现出很大的结构差异，而环的其余部分几乎没有移动，这是由于通过 Arg107 形成的盐桥相互作用。此外，发现 Arg107 与 Gln53 一起在Msm UdgX的生化特性中发挥重要作用。我们的研究为Msm UdgX 催化提供了新的见解，并提高了我们对这种独特酶的理解。