Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr 45470, Germany.
Department of Chemistry, Philipps-Universität Marburg, Marburg 35032, Germany.
Nat Commun. 2017 Mar 28;8:14876. doi: 10.1038/ncomms14876.
The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N-O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)-N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.
酸/碱催化的 5-硝基苯并异恶唑的 Kemp 消除反应生成 2-氰基-4-硝基苯酚，长期以来一直是具有非天然反应的酶的设计平台，为蛋白质科学提供了新的机制见解。在这里，我们描述了一个基于 P450-BM3 的氧化还原催化的替代概念，通过一种根本不同的机制产生相同的 Kemp 产物。QM/MM 计算表明，它涉及到底物的 N-原子与血红素-Fe(II)的配位，伴随着电子转移和同时的 N-O 异裂，释放出具有氮自由基部分 Fe(III)-N·和苯氧阴离子的中间体。产物的形成是通过键的旋转和 H 转移实现的。两个经过合理选择的点突变导致活性显著增加。结果阐明了在人类 P450 催化免疫调节剂药物来氟米特代谢中普遍存在的机制不确定性，来氟米特也同样通过 P450-BM3 发生氧化还原介导的 Kemp 消除反应。其他基于异恶唑的药物可能也通过氧化还原机制代谢。我们的工作为设计未来的人工酶提供了基础。
