As an abundantly present tobacco component, carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) has also been detected in atmospheric particulate matter, suggesting the ineluctable exposure risk of this contaminant. NNK metabolic activation by cytochrome P450 enzymes (CYPs) is a prerequisite to exerting its genotoxicity, but the metabolic regioselectivity and mechanism are still unknown. Here the binding feature and regioselectivity of CYPs 1A1, 1A2, 2A6, 2A13, 2B6, and 3A4 toward NNK are unraveled through molecular docking and molecular dynamics (MD) simulations. Binding mode analyses reveal that 1A2 and 2B6 have definite preferences for NNK α-methyl hydroxylation, while the other four CYPs preferentially catalyze α-methylene hydroxylation. The binding affinities between NNK and CYPs evaluated by the binding free energies follow the order 2A13 > 2B6 > 1A2 > 2A6 > 1A1 > 3A4. Density functional theory (DFT) calculations are further performed to characterize the mechanism of NNK biotransformation. Results show that the α-hydroxyNNK generated from α-hydroxylation may undergo nonenzymatic decomposition to form genotoxic diazohydroxide and aldehyde, and further oxidation by P450 to yield nitrosamide, which mainly contributes to NNK toxification capacity. Meanwhile the pyridine N-oxidation and denitrosation of Cα-radical intermediate play an important role in detoxifying NNK. Overall, the present study provides the molecular basis for CYP-catalyzed regioselectivity and mechanism of NNK biotransformation, which can enable the identification of metabolites for assessing the health risk of individual NNK exposure.

中文翻译：

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细胞色素P450对致癌性4-（甲基亚硝胺基）-（3-吡啶基）-1-丁酮的代谢区域选择性的分子基础和机理。

作为大量存在的烟草成分，还已经在大气颗粒物中检测到致癌物4-（甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮（NNK），这表明该污染物不可避免地具有暴露风险。细胞色素P450酶（CYP）对NNK的代谢激活是发挥其遗传毒性的先决条件，但代谢区域选择性和机制仍未知。此处通过分子对接和分子动力学（MD）模拟揭示了CYP 1A1、1A2、2A6、2A13、2B6和3A4对NNK的结合特征和区域选择性。结合模式分析显示1A2和2B6对NNKα-甲基羟基化具有明确的偏好，而其他四个CYP优先催化α-亚甲基羟基化。通过结合自由能评估的NNK和CYP之间的结合亲和力遵循2A13> 2B6> 1A2> 2A6> 1A1> 3A4的顺序。进一步执行密度泛函理论（DFT）计算以表征NNK生物转化的机制。结果表明，由α-羟基化反应生成的α-羟基NNK可能会发生非酶分解反应，形成遗传毒性重氮氢氧化物和醛，并被P450进一步氧化生成亚硝酰胺，这主要有助于NNK的毒性。同时，吡啶N-氧化和Cα-自由基中间体的脱亚硝化作用在NNK的解毒中起着重要的作用。总体而言，本研究为CYP催化的区域选择性和NNK生物转化的机理提供了分子基础，