Molecular Mechanisms of Oxygen Activation and Hydrogen Peroxide Formation in Lytic Polysaccharide Monooxygenases
Molecular Mechanisms of Oxygen Activation and Hydrogen Peroxide Formation in Lytic Polysaccharide Monooxygenases
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DOI：
10.1021/acscatal.9b00778
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发表时间：
2019-06-01
期刊：
影响因子：
12.9
通讯作者：
Rovira, Carme
中科院分区：
文献类型：
--
作者：
Wang, Binju;Walton, Paul H.;Rovira, Carme
裂解多糖单加氧酶 (LPMO) 是铜依赖性酶，用于降解难降解的多糖，例如几丁质和纤维素。与经典的水解酶（纤维素酶）不同，LPMO 通过使用氧气和还原剂的氧化机制催化糖苷键的裂解。完整的酶分子机制，从还原剂的初始电子转移到氧活化和过氧化氢形成，尚不清楚。利用量子力学/分子力学 (QM/MM) 元动力学模拟，我们发现了在抗坏血酸存在下 LPMO 的氧活化机制，抗坏血酸是 LPMO 测定中最常用的还原剂之一。我们的模拟通过从抗坏血酸中轻松提取 H 原子，捕获了 Cu(II)-O-2(-) 和 Cu(II)-OOH- 中间体的顺序形成。通过研究 Cu(II)-OOH- 中间体所有可能的反应途径，我们排除了通过 Cu(II)-OOH- 直接 O-O 裂解形成 Cu(II)-O 中心点的可能性。同时，我们发现了一条可能的途径，其中 Cu(II)-OOH- 的近端 O 原子从抗坏血酸中提取氢原子，产生 Cu(I) 和 H2O2。原位生成的 H2O2 要么通过均裂反应转化为 LPMO-Cu(II)-O 中心点，要么以非耦合路径扩散到大量水中。这两条途径的竞争强烈依赖于碳水化合物底物的结合，碳水化合物底物在阻挡原位生成的 H2O2 分子方面发挥着作用，防止其从活性位点扩散到大量水中。基于目前的结果，我们提出了与现有实验信息一致的 LPMO 催化循环。特别是，它解释了 LPMO 与 H2O2 反应性的神秘底物依赖性。
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes for the degradation of recalcitrant polysaccharides such as chitin and cellulose. Unlike classical hydrolytic enzymes (cellulases), LPMOs catalyze the cleavage of the glycosidic bond via an oxidative mechanism using oxygen and a reductant. The full enzymatic molecular mechanisms, starting from the initial electron transfer from a reductant to oxygen activation and hydrogen peroxide formation, are not yet understood. Using quantum mechanics/molecular mechanics (QM/MM) metadynamics simulations, we have uncovered the oxygen activation mechanisms by LPMO in the presence of ascorbic acid, one of the most-used reductants in LPMOs assays. Our simulations capture the sequential formation of Cu(II)-O-2(-) and Cu(II)-OOH- intermediates via facile H atom abstraction from ascorbate. By investigating all the possible reaction pathways from the Cu(II)-OOH- intermediate, we ruled out Cu(II)-O center dot- formation via direct O-O cleavage of Cu(II)-OOH-. Meanwhile, we identified a possible pathway in which the proximal O atom of Cu(II)-OOH- abstracts a hydrogen atom from ascorbate, leading to Cu(I) and H2O2. The in-situ-generated H2O2 either converts to LPMO-Cu(II)-O center dot- via a homolytic reaction, or diffuses into the bulk water in an uncoupled pathway. The competition of these two pathways is strongly dependent on the binding of the carbohydrate substrate, which plays a role in barricading the in-situ generated H2O2 molecule, preventing its diffusion from the active site into the bulk water. Based on the present results, we propose a catalytic cycle of LPMOs that is consistent with the experimental information available. In particular, it explains the enigmatic substrate dependence of the reactivity of the LPMO with H2O2.