Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Department of Chemistry, University of California Davis, Davis, CA 95616, USA.
J Mol Biol. 2021 Jul 23;433(15):167097. doi: 10.1016/j.jmb.2021.167097. Epub 2021 Jun 6.
DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2'-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.
DNA 糖苷酶通过切断 N-糖苷键来去除受损或修饰的碱基，随后通过碱基切除修复来恢复正确的核苷酸。除了切除威胁性的损伤外，DNA 糖苷酶还通过介导 DNA 去甲基化来参与表观遗传调控，并发挥其他重要功能。然而，许多糖苷酶的催化机制仍未得到很好的定义，包括 MBD4（甲基-CpG 结合域 IV），它是螺旋-发夹-螺旋（HhH）超家族的成员。MBD4 从 G·T 错配中切除胸腺嘧啶，抑制由 5-甲基胞嘧啶脱氨引起的突变，它还去除与鸟嘌呤错配的尿嘧啶和修饰的尿嘧啶（如 5-羟甲基尿嘧啶）。为了研究 MBD4 的机制，我们在催化的三个阶段解决了酶-DNA 复合物的高分辨率结构。使用不可切割的底物类似物 2'-脱氧-假尿嘧啶，我们确定了野生型 MBD4 的第一个酶-底物复合物结构，该结构证实了介导损伤识别的相互作用，并表明高度保守的 HhH 酶中的催化天冬氨酸结合了假定的亲核水分子并稳定了过渡态。观察到突变天冬氨酸（至甘氨酸）将活性降低 2700 倍表明其在催化中起重要作用，但可能不是像以前提出的那样作为双置换反应中的亲核体。与直接置换水解一致，酶-产物复合物的结构表明反应导致构型反转。含有 1-氮杂脱氧核糖的 DNA 的结构模型为潜在的氧杂碳正离子中间体，并表明天冬氨酸可以促进亲电体向亲核水的迁移。最后，这些结构提供了 HhH 基序的详细快照，告知这些普遍存在的金属结合元件如何介导 DNA 结合。
