State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
Appl Environ Microbiol. 2024 Mar 20;90(3):e0225523. doi: 10.1128/aem.02255-23. Epub 2024 Feb 28.
Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCE S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.
黄素蛋白单加氧酶催化反应，包括羟化和环氧化，参与天然底物和工业污染物的分解代谢、解毒和生物合成。其中，S16 中的 6-羟基-3-琥珀酰吡啶（HSP）单加氧酶（HspB）促进尼古丁吡啶环的羟化和 C-C 键断裂。然而，生物降解的机制仍然难以捉摸。在这里，我们细化了 HspB 的晶体结构，并阐明了氧化羟化和 C-C 断裂过程背后的详细机制。利用关于结合辅因子黄素腺嘌呤二核苷酸（FAD）和 HSP 底物的结构域的结构信息，我们使用分子动力学模拟和量子/分子力学计算来证明，从反应性 FAD 过氧化物物种（C4a-过氧黄素）向 HSP 底物的 C3 原子转移一个氧原子是一个限速步骤，计算出的反应势垒约为 20 kcal/mol。随后，氢原子反弹回 FAD 辅因子，形成 C4a-羟基黄素。然后残基半胱氨酸 218 催化随后的 C-C 断裂水解过程。我们的研究结果有助于更深入地了解黄素蛋白在吡啶的自然转化和尼古丁降解中 HspB 的多种功能。
重要性：S16 在降解尼古丁（一种具有显著环境挑战的有毒吡啶衍生物）方面发挥着关键作用。这项研究强调了一种关键酶 HspB（6-羟基-3-琥珀酰吡啶单加氧酶）通过吡咯烷途径分解尼古丁。HspB 利用分子氧和黄素腺嘌呤二核苷酸辅因子将底物的侧链羟化并断裂。细化的 HspB 晶体结构的结构分析，结合最先进的计算，揭示了其独特的机制。半胱氨酸 218 的关键作用在其同源酶中从未被发现。我们的发现不仅加深了我们对细菌尼古丁降解的理解，也为环境清理和药物开发的应用开辟了道路。
