A redox-mediated Kemp eliminase

Aitao Li, Binju Wang, Adriana Ilie, Kshatresh D. Dubey, Gert Bange, Ivan V. Korendovych, Sason Shaik, Manfred T. Reetz

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


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.

Original languageEnglish (US)
Article number14876
JournalNature Communications
StatePublished - Mar 28 2017

ASJC Scopus subject areas

  • General Chemistry
  • General Biochemistry, Genetics and Molecular Biology
  • General Physics and Astronomy


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