Dioxygen Activation and Nδ,Nε-Dihydroxylation Mechanism Involved in the Formation of N-Nitrosourea Pharmacophore in Streptozotocin Catalyzed by Nonheme Diiron Enzyme SznF,Inorganic Chemistry

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Dioxygen Activation and Nδ,Nε-Dihydroxylation Mechanism Involved in the Formation of N-Nitrosourea Pharmacophore in Streptozotocin Catalyzed by Nonheme Diiron Enzyme SznF
Inorganic Chemistry ( IF 4.3 ) Pub Date : 2022-09-23 , DOI: 10.1021/acs.inorgchem.2c02814
Yijing Wang ₁ , Lihua Dong ₂ , Hao Su ₃ , Yongjun Liu ₁

Affiliation

SznF is a nonheme diiron-dependent enzyme that catalyzes the critical N-nitrosation involved in the formation of the N-nitrosourea moiety in the pancreatic cancer drug streptozotocin. The N-nitrosation contains two successive N-hydroxylation and N-nitrosation steps, which are carried out by two separate active sites, namely, the central domain and cupin domain. Recently, the crystal structure of SznF was obtained, and the central domain was proved to contain a diiron cofactor to catalyze the N-hydroxylation. In this work, to gain insights into the O₂ activation and the successive N-hydroxylation mechanism, on the basis of the high-resolution crystal structure, the enzyme–substrate complex models were constructed, and a series of combined QM/MM calculations were performed. Based on our calculations, the activation of O₂ starts from the diiron(II,III)-superoxo (S) to generate the diiron(IV)-oxo species (Q) via a diiron(III,III)-peroxo (P)-like transition state or unstable intermediate (P′), and species P′ can be described as a hybridization of diiron(IV)-oxo species and diiron(III,III)-peroxo (P) owing to the long distances of Fe1-Fe2 (4.22 Å) and O1–O2 (1.89 Å), which is different from those of other nonheme diiron enzymes. In the following hydroxylation of N^δ and N^ε, the N^δ-hydroxylation was confirmed to occur first, agreeing with the experimental observations. Because the diiron(IV)-oxo species (Q) is responsible for hydroxylation, the reaction follows the H-abstraction/OH rebound mechanism, and the first abstraction occurs on the N^δ–H rather than N^ε–H, which may be attributed to the different orientation of Fe(IV)-oxo relative to N–H as well as the bond dissociation enthalpies of two N–H bonds. The hydroxylation of N-methyl-L-arginine does not employ the diiron(III,III)-hydroperoxo (P″) to trigger the electrophilic attack of the guanidine to directly form the N–O bond, as previously suggested. In addition, our calculations also revealed that the direct attack of the Fe(IV)═O unit to the N^δ of the substrate corresponds to a higher barrier than that in the H-abstraction/OH rebound mechanism. These results may provide useful information for understanding the formation of the di-hydroxylation intermediate involved in the biosynthesis of N-nitrosation.

中文翻译：

非血红素二铁酶 SznF 催化链脲佐菌素形成 N-亚硝基脲药效团的分子氧活化及 Nδ,Nε-二羟基化机制

SznF 是一种非血红素二铁依赖性酶，可催化胰腺癌药物链脲佐菌素中 N-亚硝基脲部分形成所涉及的关键 N-亚硝化。N-亚硝化包含两个连续的 N-羟基化和 N-亚硝化步骤，这些步骤由两个独立的活性位点进行，即中心域和 Cupin 域。最近，获得了 SznF 的晶体结构，证明中心域含有二铁辅因子来催化 N-羟基化。在这项工作中，为了深入了解 O ₂活化和相继的N-羟基化机理，在高分辨率晶体结构的基础上，构建了酶-底物复合物模型，并进行了一系列的QM/MM组合计算。根据我们的计算，O ₂的活化从二铁(II,III)-超氧(S)开始，通过二铁(III,III)-过氧(P)生成二铁(IV)-氧(Q)类过渡态或不稳定中间体（P'），由于 Fe1- 的长距离，物种 P' 可以描述为 diiron(IV)-oxo 物种和 diiron(III,III)-peroxo(P) 的杂化Fe2 (4.22 Å) 和 O1-O2 (1.89 Å)，与其他非血红素二铁酶不同。在以下 N ^δ和 N ^ε的羟基化中，N ^δ确认首先发生-羟基化，与实验观察结果一致。因为二铁（IV）-氧代物种（Q）负责羟基化，所以反应遵循 H-抽象/OH 反弹机制，第一个抽象发生在 N ^δ -H 而不是 N ^ε -H 上，这可能是归因于 Fe(IV)-oxo 相对于 N-H 的不同取向以及两个 N-H 键的键解离焓。如前所述， N-甲基-L-精氨酸的羟基化不使用二铁（III，III）-氢过氧（P”）来触发胍的亲电攻击以直接形成N-O键。此外，我们的计算还揭示了 Fe(IV)=O 单元对 N ^{δ的直接攻击。}与 H-抽象/OH 回弹机制相比，底物的势垒对应于更高的势垒。这些结果可能为了解参与 N-亚硝化生物合成的二羟基化中间体的形成提供有用的信息。

更新日期：2022-09-23

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