E. coli AlkB and human ALKBH2 belong to the AlkB family enzymes, which contain several α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases that repair alkylated DNA. Specifically, the AlkB enzymes catalyze decarboxylation of α-KG to generate a high-valent Fe(IV)-oxo species that oxidizes alkyl groups on DNA adducts. AlkB and ALKBH2 have been reported to differentially repair select etheno adducts, with preferences for 1,N⁶-ethenoadenine (1,N⁶-εA) and 3,N⁴-ethenocytosine (3,N⁴-εC) over 1,N²-ethenoguanine (1,N²-εG). However, N²,3-ethenoguanine (N²,3-εG), the most common etheno adduct, is not repaired by the AlkB enzymes. Unfortunately, a structural understanding of the differential activity of E. coli AlkB and human ALKBH2 is lacking due to challenges acquiring atomistic details for a range of substrates using experiments. This study uses both molecular dynamics (MD) simulations and ONIOM(QM:MM) calculations to determine how the active site changes upon binding each etheno adduct and characterizes the corresponding catalytic impacts. Our data reveal that the preferred etheno substrates (1,N⁶-εA and 3,N⁴-εC) form favorable interactions with catalytic residues that situate the lesion near the Fe(IV)-oxo species and permit efficient oxidation. In contrast, although the damage remains correctly aligned with respect to the Fe(IV)-oxo moiety, repair of 1,N²-εG is mitigated by increased solvation of the active site and a larger distance between Fe(IV)-oxo and the aberrant carbons. Binding of non-substrate N²,3-εG in the active site disrupts key DNA–enzyme interactions, and positions the aberrant carbon atoms even further from the Fe(IV)-oxo species, leading to prohibitively high barriers for oxidative catalysis. Overall, our calculations provide the first structural insight required to rationalize the experimentally-reported substrate specificities of AlkB and ALKBH2 and thereby highlight the roles of several active site residues in the repair of etheno adducts that directly correlates with available experimental data. These proposed catalytic strategies can likely be generalized to other α-KG/Fe(II)-dependent dioxygenases that play similar critical biological roles, including epigenetic and post-translational regulation.

大肠杆菌AlkB 和人 ALKBH2 属于 AlkB 家族酶，其中包含多种 α-酮戊二酸 (α-KG)/Fe(II) 依赖性双加氧酶，可修复烷基化 DNA。具体而言，AlkB 酶催化 α-KG 的脱羧，生成高价 Fe(IV)-氧代物质，该物质可氧化 DNA 加合物上的烷基。据报道，AlkB 和 ALKBH2 对选择的乙烯加合物进行差异修复，优先选择 1, N ⁶ -乙烯腺嘌呤(1,N ⁶ -εA) 和 3, N ^{4 -}乙烯胞嘧啶 (3,N ⁴ -εC) 超过 1, N ² -乙烯鸟嘌呤 (1,N ² -εG)。然而，N ² ,3-乙烯鸟嘌呤（N ²,3-εG) 是最常见的乙烯加合物，不会被 AlkB 酶修复。不幸的是，由于使用实验获取一系列底物的原子细节的挑战，缺乏对大肠杆菌AlkB 和人类 ALKBH2差异活性的结构理解。本研究使用分子动力学 (MD) 模拟和 ONIOM(QM:MM) 计算来确定活性位点在结合每个乙烯加合物时如何变化，并表征相应的催化影响。我们的数据表明，优选的乙烯底物（1,N ⁶ -εA 和 3,N ⁴-εC) 与催化残基形成有利的相互作用，催化残基位于 Fe(IV)-oxo 物种附近并允许有效氧化。相比之下，尽管损伤与 Fe(IV)-oxo 部分保持正确对齐，但 1,N ² -εG 的修复通过增加活性位点的溶剂化和 Fe(IV)-oxo 和异常的碳。非底物 N ^{2 的结合}活性位点中的 ,3-εG 破坏了关键的 DNA-酶相互作用，并将异常碳原子定位在离 Fe(IV)-oxo 更远的位置，导致氧化催化的障碍过高。总体而言，我们的计算提供了合理化实验报告的 AlkB 和 ALKBH2 底物特异性所需的第一个结构洞察力，从而突出了几个活性位点残基在修复与可用实验数据直接相关的乙烯加合物中的作用。这些提议的催化策略可能会推广到其他 α-KG/Fe(II) 依赖性双加氧酶，这些酶具有类似的关键生物学作用，包括表观遗传和翻译后调节。