Catalytic Mechanism of Nogalamycin Monoxygenase: How Does Nature Synthesize Antibiotics without a Metal Cofactor?
Catalytic Mechanism of Nogalamycin Monoxygenase: How Does Nature Synthesize Antibiotics without a Metal Cofactor?
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DOI：
10.1021/acs.jpcb.8b09648
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发表时间：
2018-11
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影响因子：
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通讯作者：
Fabián G Cantú Reinhard;J. DuBois;S. D. de Visser
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作者：
Fabián G Cantú Reinhard;J. DuBois;S. D. de Visser
诺加霉素单加氧酶（NMO）是一个酶家族的成员，它催化四环素抗生素生物合成的关键步骤，四环素抗生素用于治疗人类乳腺癌，使用分子氧进行底物氧化，但没有明显的辅助因子。由于大多数单加氧酶和双加氧酶都含有过渡金属中心（Fe/Cu）或黄素，这就引出了NMO如何催化这种不寻常的氧原子从分子氧直接转移到底物的问题。我们利用密度泛函理论和量子力学/分子力学对NMO的机理和催化循环进行了详细的计算研究。我们考虑了不同质子化状态下的底物及其与氧化剂O2和O2-•通过电子转移、质子转移或氢原子转移的反应。本文所述模型的最低能量途径是中性底物与超氧阴离子自由基（O2-•）的反应。然而，在没有可用的自由超氧阴离子的情况下，3O2和底物之间的替代中性途径在室温下是可以达到的，尽管势垒的能量要高约20千卡摩尔-1，因此反应会慢得多。与之前酶促反应和非催化反应的实验结果相反，底物在去质子化状态下的反应机制具有高能量，因此提出了机制建议。热力学分析表明，底物有一个非常弱的C-H键，可以被弱氧化剂激活，因此，金属辅因子可能不需要氧化这种特殊的底物。最后，研究了位点定向突变，其中活性位点Asn残基被替换，这些残基在将氧气引导到底物的c12位置上的功能得到了强调。总的来说，NMO表现出一种多用途的反应模式，其中底物可以通过几种低能途径与氧化剂和处于不同氧化和质子化状态的底物一起被激活。
Nogalamycin monoxygenase (NMO) is a member of a family of enzymes that catalyze a key step in the biosynthesis of tetracycline antibiotics used to treat, for example, breast cancer in humans, using molecular oxygen for substrate oxidation but without an apparent cofactor. As most monoxygenases and dioxygenases contain a transition metal center (Fe/Cu) or flavin, this begs the question how NMO catalyzes this unusual oxygen atom transfer reaction from molecular oxygen to substrate directly. We performed a detailed computational study on the mechanism and catalytic cycle of NMO using density functional theory and quantum mechanics/molecular mechanics on the full protein. We considered the substrate in various protonation states and its reaction with oxidant O2 as well as O2-• through either electron transfer, proton transfer, or hydrogen atom transfer. The lowest energy pathway for the models presented here is a reaction of the neutral substrate with a superoxo anion radical (O2-•). In the absence of available free superoxo anions, however, the alternative neutral pathway between 3O2 and the substrate may be accessible at room temperature, although the barrier is higher in energy by about 20 kcal mol-1 and therefore the reaction will be much slower. In contrast to previous experimental findings for both the enzymatic and uncatalyzed reactions, the mechanisms with the substrate in its deprotonated state were found to be high in energy, and therefore mechanistic suggestions are proposed. A thermodynamic analysis shows that the substrate has a very weak C-H bond that can be activated by a weak oxidant, and hence, a metal cofactor may not be needed for oxidizing this particular substrate. Finally, site-directed mutations were studied where active-site Asn residues were replaced, and the function of these residues in guiding oxygen to the C12-position of the substrate was highlighted. Overall, NMO shows a versatile reactivity pattern, where the substrate can be activated by several low-energy pathways with oxidants and substrates in various oxidation and protonation states.